Compositions for treating spinal muscular atrophy

ABSTRACT

The present invention provides pharmaceutical compositions comprising a compound of formula (I) 
     
       
         
         
             
             
         
       
         
         
           
             wherein A, R 1 , R 2  and R 3  are as described herein, as well as pharmaceutically acceptable salts thereof. Further the present invention is concerned with the manufacture of the pharmaceutical compositions comprising a compound of formula (I) and their use as medicaments.

INTRODUCTION

The present invention provides pharmaceutical compositions comprisingcompounds which are SMN2 gene splicing modulators, their manufacture andtheir use for the treatment, delay of progression or amelioration ofspinal muscular atrophy (SMA). Further, the pharmaceutical compositionsof the invention may optionally comprise cytoprotectors. The inventionfurther relates to the combined use of SMN2 gene splicing modulators andcytoprotectors for use in the treatment or amelioration of spinalmuscular atrophy (SMA).

In particular, the present invention relates to pharmaceuticalcompositions comprising compounds of formula (I)

wherein A, R¹, R² and R³ are as described herein, and pharmaceuticallyacceptable salts thereof.

BACKGROUND

Spinal muscular atrophy (SMA), in its broadest sense, describes acollection of inherited and acquired central nervous system (CNS)diseases characterized by progressive motor neuron loss in the spinalcord and brainstem causing muscle weakness and muscle atrophy. The mostcommon form of SMA is caused by mutations in the Survival Motor Neuron(SMN) gene and manifests over a wide range of severity affecting infantsthrough adults (Crawford and Pardo, Neurobiol. Dis., 1996, 3:97).

Infantile SMA is the most severe form of this neurodegenerativedisorder. Symptoms include muscle weakness, poor muscle tone, weak cry,limpness or a tendency to flop, difficulty sucking or swallowing,accumulation of secretions in the lungs or throat, feeding difficulties,and increased susceptibility to respiratory tract infections. The legstend to be weaker than the arms and developmental milestones, such aslifting the head or sitting up, cannot be reached. In general, theearlier the symptoms appear, the shorter the lifespan. As the motorneuron cells deteriorate, symptoms appear shortly afterward. The severeforms of the disease are fatal and all forms have no known cure. Thecourse of SMA is directly related to the rate of motor neuron celldeterioration and the resulting severity of weakness. Infants with asevere form of SMA frequently succumb to respiratory disease due toweakness in the muscles that support breathing. Children with milderforms of SMA live much longer, although they may need extensive medicalsupport, especially those at the more severe end of the spectrum. Theclinical spectrum of SMA disorders has been divided into the followingfive groups.

-   -   (a) Type 0 SMA (In Utero SMA) is the most severe form of the        disease and begins before birth. Usually, the first symptom of        Type 0 SMA is reduced movement of the fetus that can first be        observed between 30 and 36 weeks of pregnancy. After birth,        these newborns have little movement and have difficulties with        swallowing and breathing.    -   (b) Type 1 SMA (Infantile SMA or Werdnig-Hoffmann disease)        presents symptoms between 0 and 6 months. form of SMA is also        very severe. Patients never achieve the ability to sit, and        death usually occurs within the first 2 years without        ventilatory support.    -   (c) Type 2 SMA (Intermediate SMA) has an age of onset at 7-18        months. Patients achieve the ability to sit unsupported, but        never stand or walk unaided. Prognosis in this group is largely        dependent on the degree of respiratory involvement.    -   (d) Type 3 SMA (Juvenile SMA or Kugelberg-Welander disease) is        generally diagnosed after 18 months. Type 3 SMA individuals are        able to walk independently at some point during their disease        course but often become wheelchair-bound during youth or        adulthood.    -   (e) Type 4 SMA (Adult onset SMA). Weakness usually begins in        late adolescence in the tongue, hands, or feet, then progresses        to other areas of the body. The course of adult SMA is much        slower and has little or no impact on life expectancy.

The SMN gene has been mapped by linkage analysis to a complex region inchromosome 5q. In humans, this region contains an approximately 500thousand base pairs (kb) inverted duplication resulting in two nearlyidentical copies of the SMN gene. SMA is caused by an inactivatingmutation or deletion of the telomeric copy of the gene (SMN1) in bothchromosomes, resulting in the loss of SMN1 gene function. However, allpatients retain the centromeric copy of the gene (SMN2), and the copynumber of the SMN2 gene in SMA patients generally correlates inverselywith the disease severity; i.e., patients with less severe SMA have morecopies of SMN2. Nevertheless, SMN2 is unable to compensate completelyfor the loss of SMN1 function due to alternative splicing of exon 7caused by a translationally silent C to T mutation in exon 7. As aresult, the majority of transcripts produced from SMN2 lack exon 7 (Δ7SMN2), and encode a truncated SMN protein that has an impaired functionand is rapidly degraded.

The SMN protein is thought to play a role in RNA processing andmetabolism, having a well characterized function of mediating theassembly of a specific class of RNA-protein complexes termed snRNPs. SMNmay have other functions in motor neurons, however its role inpreventing the selective degeneration of motor neurons is not wellestablished.

In most cases, SMA is diagnosed based on clinical symptoms and by thepresence of at least one copy of the SMN1 gene test. However, inapproximately 5% of cases SMA is caused by mutation in genes other thanthe inactivation of SMN 1, some known and others not yet defined. Insome cases, when the SMN 1 gene test is not feasible or does not showany abnormality, other tests such as an electromyography (EMG) or musclebiopsy may be indicated.

Medical care for SMA patients at present is limited to supportivetherapy including respiratory, nutritional and rehabilitation care;there is no drug known to address the underlying cause of the disease.Current treatment for SMA consists of prevention and management of thesecondary effects of chronic motor unit loss. The major management issuein Type 1 SMA is the prevention and early treatment of pulmonaryproblems, which are the cause of death in the majority of the cases.While some infants afflicted with SMA grow to be adults, those with Type1 SMA have a life expectancy of less than two years.

Several mouse models of SMA have been developed. In particular, the SMNdelta exon 7 (Δ7 SMN) model (Le et al., Hum. Mol. Genet., 2005, 14:845)carries both the SMN2 gene and several copies of the Δ7 SMN2 cDNA andrecapitulates many of the phenotypic features of Type 1 SMA. The Δ7 SMNmodel can be used for both SMN2 expression studies as well as theevaluation of motor function and survival. The C/C-allele mouse model(Jackson Laboratory strain #008714, The Jackson Laboratory, Bar Harbor,Me.) provides a less severe SMA disease model, with mice having reducedlevels of both SMN2 full length (FL SMN2) mRNA and SMN protein. TheC/C-allele mouse phenotype has the SMN2 gene and a hybrid mSMN1-SMN2gene that undergoes alternative splicing, but does not have overt muscleweakness. The C/C-allele mouse model is used for SMN2 expressionstudies.

As a result of improved understanding of the genetic basis andpathophysiology of SMA, several strategies for treatment have beenexplored, but none have yet demonstrated success in the clinic.

Gene replacement of SMN1, using viral delivery vectors, and cellreplacement, using differentiated SMN1^(+/+) stem cells, havedemonstrated efficacy in animal models of SMA. More research is neededto determine the safety and immune response and to address therequirement for the initiation of treatment at the neonatal stage beforethese approaches can be applied to humans.

Correction of alternative splicing of SMN2 in cultured cells has alsobeen achieved using synthetic nucleic acids as therapeutic agents: (i)antisense oligonucleotides that target sequence elements in SMN2pre-mRNA and shift the outcome of the splicing reaction toward thegeneration of full length SMN2 mRNA (Passini et al., Sci. Transl. Med.,2011, 3: 72ra18; and, Hua et al., Nature, 2011, 478:123) and (ii)trans-splicing RNA molecules that provide a fully functional RNAsequence that replace the mutant fragment during splicing and generate afull length SMN1 mRNA (Coady and Lorson, J Neurosci., 2010, 30:126).

Other approaches under exploration include searching for drugs thatincrease SMN levels, enhance residual SMN function, or compensate forits loss. Aminoglycosides have been shown to enhance expression of astabilized SMN protein produced from Δ7 SMN2 mRNA by promoting thetranslational read-through of the aberrant stop codon, but have poorcentral nervous system penetration and are toxic after repeat dosing.Chemotherapeutic agents, such as aclarubicin, have been shown toincrease SMN protein in cell culture; however, the toxicity profile ofthese drugs prohibits long-term use in SMA patients. Some drugs underclinical investigation for the treatment of SMA include transcriptionactivators such as histone deacetylase (“HDAC”) inhibitors (e.g.,butyrates, valproic acid, and hydroxyurea), and mRNA stabilizers (mRNAdecapping inhibitor RG3039 from Repligen), the goal being to increasethe amount of total RNA transcribed from the SMN2 gene. However, the useof the HDAC inhibitors or mRNA stabilizers does not address theunderlying cause of SMA and may result in a global increase intranscription and gene expression with potential safety problems inhumans.

In an alternative approach, cytoprotective agents such as Olesoxime havebeen chosen for investigation. Such strategies are not aimed at SMN forthe treatment of SMA, but instead have been developed to protect notonly the SMN-deficient motor neurons from neurodegeneration, but alsoother systems affected by the disease, such as muscle cells. Olesoximehas shown clinical efficacy in the treatment of SMA Type 2 (IntermediateSMA) and SMA Type 3 (Juvenile SMA, non-ambulatory).

A system designed for identifying compounds that increase the inclusionof exon 7 of SMN into RNA transcribed from the SMN2 gene and certainbenzooxazole and benzoisoxazole compounds identified thereby have beendescribed in International Patent Application WO2009/151546A1. A systemdesigned for identifying compounds that cause ribosomal frameshifting toproduce a stabilized SMN protein from Δ7 SMN2 mRNA and certainisoindolinone compounds identified thereby have been described inInternational Patent Applications WO2010/019236A1 and WO2013/119916A2.

Olesoxime (Cholest-4-en-3-one oxime,(EZ)—N-(cholest-4-en-3-ylidene)hydroxylamine, CAS Registry Number22033-87-0) is a cytoprotective drug that has been found to promote thefunction and survival of neurons and other cell types underdisease-relevant stress conditions through interactions with themitochondrial permeability transition pore (mPTP).

WO 20047082581 (A2) describes the use of olesoxime for providingneuroprotection in a patient and WO 2008/142231 (A2) describespharmaceutical compositions comprising it.

Methods of synthesizing oximes of d6-cholestenons and olesoxime havebeen described e.g. by Nobel prize laureate Adolf Friedrich JohannButenandt (Butenandt A. et al., Berichte der Deutschen ChemischenGesellschaft (1936), 69B, 882-8) or by Ponsold K. et al. (Journal fuerPraktische Chemie (1964), 23(3-4), 173-6).

Mode of action of olesoxime, its toxicity, metabolism, andpharmacodynamics have been described in Martin L. J. (IDrugs (2010)13(8):568-80). In vivo, olesoxime rescues motor neurons from cell deathinduced by nerve lesion in neonatal rats and promotes nerve regenerationfollowing a nerve crush in adult mice. By promoting both axonalregeneration and survival of motor neurons, olesoxime is a rationaltherapeutic approach for SMA. Additionally, there is evidence offunctional improvement in nonclinical models of SMA.

At the molecular level, binding data indicated that olesoxime interactswith two outer mitochondrial membrane proteins which appears to modulatethe opening of the mitochondrial permeability transition pore complex(mPTP). By binding to these proteins, olesoxime may preserve essentialmitochondrial functions, such as calcium buffering in stressed neurons,thereby reducing cellular degeneration and death. The cytoprotectiveeffects were observed in primary neurons subjected to physiologicalstress, in primary cardiomyocytes subjected to anthracycline toxicityand also in mouse hepatocytes submitted to Fas-induced apoptosis. Thus,olesoxime has the potential to reduce pathological, stress-induced,apoptosis in neuronal as well as non-neuronal cells. Therefore, thesebinding sites may play a role in the neuro-, cell- and tissue-protectiveaction of olesoxime since stress-induced mitochondrial dysfunction hasbeen implicated in most neurodegenerative diseases. In vivo, the motorneuron rescue and the nerve regeneration promotion observed witholesoxime treatment confirm that olesoxime acts both at the motor neuroncell body level, at the axonal level, and potentially has a protectiveeffect on muscle (Pathak D et al. J Biological Chemistry (2015)290(37):22325-36).

Olesoxime has been developed for the treatment of Type 2 and Type 3 SMA.The clinical development program of olesoxime aimed at demonstratingmaintenance of motor function over an observation period of two years.The clinical development of olesoxime in SMA includes two clinicalstudies, a Phase Ib PK and safety study (TRO19622CLEQ1115-1) using ahard capsule formulation, and a Phase II study (TRO19622CLEQ1275-1)using an oral suspension formulation. The placebo-controlled Phase IIstudy (TRO19622CLEQ1275-1) is currently the largest and longest clinicalstudy to have been conducted for this indication. The study showedmaintenance of motor function over the two-year treatment period in theolesoxime arm compared with an approximately two-point decline in theprimary endpoint (motor function measure [MFM]) in the placebo arm,which is in keeping with the reported natural disease progression(Vuillerot C et al. Arch Phys Med Rehabil (2013) 94(8):1555-61.). ThePhase II study demonstrated a positive benefit-risk profile forolesoxime for the treatment of patients with Types 2 and 3 SMA.

Despite the progress made in understanding the genetic basis andpathophysiology of SMA, there remains a need to identify compounds andcombinations of compounds and suitable forms of administration thereofthat alter the course of spinal muscular atrophy, one of the mostdevastating childhood neurological diseases.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

The nomenclature used in this Application is based on IUPAC systematicnomenclature, unless indicated otherwise.

Any open valency appearing on a carbon, oxygen, sulfur or nitrogen atomin the structures herein indicates the presence of a hydrogen, unlessindicated otherwise.

The definitions described herein apply irrespective of whether the termsin question appear alone or in combination. It is contemplated that thedefinitions described herein can be appended to form chemically-relevantcombinations, such as e.g. “heterocycloalkylaryl”,“haloalkylheteroaryl”, “arylalkylheterocycloalkyl”, or “alkoxyalkyl”.The last member of the combination is the radical which is binding tothe rest of the molecule. The other members of the combination areattached to the binding radical in reversed order in respect of theliteral sequence, e.g. the combination amino-C₁₋₇-alkyl refers to aC₁₋₇-alkyl which is substituted by amino, or e.g. the combinationarylalkylheterocycloalkyl refers to a heterocycloalkyl-radical which issubstituted by an alkyl which is substituted by an aryl.

The term “moiety” refers to an atom or group of chemically bonded atomsthat is attached to another atom or molecule by one or more chemicalbonds thereby forming part of a molecule. For example, the variables A,R¹, R² and R³ of formula (I) refer to moieties that are attached to thecore structure of formula (I) by a covalent bond.

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogens by substituents.

The term “optional” or “optionally” denotes that a subsequentlydescribed event or circumstance can but need not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not.

The term “substituent” denotes an atom or a group of atoms replacing ahydrogen atom on the parent molecule.

The term “substituted” denotes that a specified group bears one or moresubstituents. Where any group can carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The terms “compound(s) of this invention” and “compound(s) of thepresent invention” refer to compounds as disclosed herein andstereoisomers, tautomers, solvates, and salts (e.g., pharmaceuticallyacceptable salts) thereof.

When the compounds of the invention are solids, it is understood bythose skilled in the art that these compounds, and their solvates andsalts, may exist in different solid forms, particularly differentcrystal forms, all of which are intended to be within the scope of thepresent invention and specified formulae.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, and polyamine resins.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. In describing an optically active compound, the prefixes Dand L, or R and S, are used to denote the absolute configuration of themolecule about its chiral center(s). The substituents attached to thechiral center under consideration are ranked in accordance with theSequence Rule of Cahn, Ingold and Prelog. (Cahn et al. Angew. Chem.Inter. Edit. 1966, 5, 385; errata 511). The prefixes D and L or (+) and(−) are employed to designate the sign of rotation of plane-polarizedlight by the compound, with (−) or L designating that the compound islevorotatory. A compound prefixed with (+) or D is dextrorotatory.

The term “chiral center” denotes a carbon atom bonded to fournonidentical substituents. The term “chiral” denotes the ability ofnon-superimposability with the mirror image, while the term “achiral”refers to embodiments which are superimposable with their mirror image.Chiral molecules are optically active, i.e., they have the ability torotate the plane of plane-polarized light.

Compounds of the present invention can have one or more chiral centersand can exist in the form of optically pure enantiomers, mixtures ofenantiomers such as, for example, racemates, optically purediastereoisomers, mixtures of diastereoisomers, diastereoisomericracemates or mixtures of diastereoisomeric racemates. Whenever a chiralcenter is present in a chemical structure, it is intended that allstereoisomers associated with that chiral center are encompassed by thepresent invention.

The terms “halo”, “halogen”, and “halide” are used interchangeablyherein and denote fluoro, chloro, bromo, or iodo. One particular exampleof halogen is fluoro.

The term “alkyl” denotes a monovalent linear or branched saturatedhydrocarbon group of 1 to 12 carbon atoms. In particular embodiments,alkyl has 1 to 7 carbon atoms, and in more particular embodiments 1 to 4carbon atoms. Examples of alkyl include methyl, ethyl, propyl,isopropyl, n-butyl, iso-butyl, sec-butyl, or tert-butyl. Particularexamples for alkyl are methyl and ethyl.

The term “haloalkyl” denotes an alkyl group wherein at least one of thehydrogen atoms of the alkyl group has been replaced by same or differenthalogen atoms, particularly fluoro atoms. Examples of haloalkyl includemonofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, forexample 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl,fluoromethyl, or trifluoromethyl and the like. The term “perhaloalkyl”denotes an alkyl group where all hydrogen atoms of the alkyl group havebeen replaced by the same or different halogen atoms.

The term “bicyclic ring system” denotes two rings which are fused toeach other via a common single or double bond (annelated bicyclic ringsystem), via a sequence of three or more common atoms (bridged bicyclicring system) or via a common single atom (spiro bicyclic ring system).Bicyclic ring systems can be saturated, partially unsaturated,unsaturated or aromatic. Bicyclic ring systems can comprise heteroatomsselected from N, O and S.

The term “cycloalkyl” denotes a saturated monocyclic or bicyclichydrocarbon group of 3 to 10 ring carbon atoms. In particularembodiments cycloalkyl denotes a monovalent saturated monocyclichydrocarbon group of 3 to 8 ring carbon atoms. Bicyclic means consistingof two saturated carbocycles having one or more carbon atoms in common.Particular cycloalkyl groups are monocyclic. Examples for monocycliccycloalkyl are cyclopropyl, cyclobutanyl, cyclopentyl, cyclohexyl orcycloheptyl. Examples for bicyclic cycloalkyl arebicyclo[2.2.1]heptanyl, or bicyclo[2.2.2]octanyl. One particular exampleof cycloalkyl is cyclopropyl.

The term “heterocycloalkyl” denotes a saturated or partly unsaturatedmono-, bi- or tricyclic ring system of 3 to 9 ring atoms, comprising 1,2, or 3 ring heteroatoms selected from N, O and S, the remaining ringatoms being carbon. In particular embodiments, heterocycloalkyl is amonovalent saturated monocyclic ring system of 4 to 7 ring atoms,comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, theremaining ring atoms being carbon. Examples for monocyclic saturatedheterocycloalkyl are aziridinyl, oxiranyl, azetidinyl, oxetanyl,pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl,imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl,piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl,morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl,diazepanyl, homopiperazinyl, or oxazepanyl. Examples for bicyclicsaturated heterocycloalkyl are 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl,8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl,3-oxa-9-aza-bicyclo[3.3.1]nonyl, or 3-thia-9-aza-bicyclo[3.3.1]nonyl.Examples of a partly unsaturated heterocycloalkyl are dihydrofuryl,imidazolinyl, dihydro-oxazolyl, tetrahydro-pyridinyl, or dihydropyranyl.Particular examples of heterocycloalkyl are 1,4-diazepanyl,hexahydropyrrolo[1,2-a]pyrazinyl, piperidinyl, piperazinyl andpyrrolidinyl. More particular examples of heterocycloalkyl arehexahydropyrrolo[1,2-a]pyrazinyl and piperazinyl.

The term “N-heterocycloalkyl” denotes a heterocycloalkyl radicalcontaining at least one nitrogen ring atom and where the point ofattachment of the heterocycloalkyl radical to the rest of the moleculeis through a nitrogen ring atom. Particular examples ofN-heterocycloalkyl are 1,4-diazepanyl, hexahydropyrrolo[1,2-a]pyrazinyl,piperidinyl, piperazinyl and pyrrolidinyl. More particular examples ofN-heterocycloalkyl are hexahydropyrrolo[1,2-a]pyrazinyl and piperazinyl.

The term “basicity” in reference to a compound is expressed herein bythe negative decadic logarithm of the acidity constant of the conjugateacid (pKa=-log Ka). The larger the pKa of the conjugate acid, thestronger the base (pKa+pKb=14). In this application, an atom orfunctional group is denoted “basic” if it is suitable to accept a protonand if the calculated pKa of its conjugate acid is at least 7, moreparticularly if the calculated pKa of its conjugate acid is at least7.8, most particularly if the calculated pKa of its conjugate acid is atleast 8. pKa values were calculated in-silico as described in F.Milletti et al., J. Chem. Inf. Model (2007) 47:2172-2181.

The term “alkylene” denotes a linear saturated divalent hydrocarbongroup of 1 to 7 carbon atoms or a divalent branched saturatedhydrocarbon group of 3 to 7 carbon atoms. Examples of alkylene groupsinclude methylene, ethylene, propylene, 2-methylpropylene, butylene,2-ethylbutylene, pentylene, hexylene. Particular examples for alkyleneare ethylene, propylene, and butylene.

The term “amino” denotes a group of the formula —NR′R″ wherein R′ and R″are independently hydrogen, alkyl, alkoxy, cycloalkyl, heterocycloalkyl,aryl, heteroaryl or as described herein. Alternatively, R′ and R″,together with the nitrogen to which they are attached, can form aheterocycloalkyl. The term “primary amino” denotes a group wherein bothR′ and R″ are hydrogen. The term “secondary amino” denotes a groupwherein R′ is hydrogen and R″ is a group other than hydrogen. The term“tertiary amino” denotes a group wherein both R′ and R″ are other thanhydrogen. Particular secondary and tertiary amines are methylamine,ethylamine, propylamine, isopropylamine, phenylamine, benzylaminedimethylamine, diethylamine, dipropylamine and diisopropylamine.

The term “active pharmaceutical ingredient” (or “API”) denotes thecompound or molecule in a pharmaceutical composition that has aparticular biological activity.

The terms “pharmaceutical composition” and “pharmaceutical formulation”(or “formulation”) are used interchangeably and denote a mixture orsolution comprising a therapeutically effective amount of an activepharmaceutical ingredient together with pharmaceutically acceptableexcipients to be administered to a mammal, e.g., a human in needthereof.

The term “pharmaceutically acceptable” denotes an attribute of amaterial which is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic, and neither biologically nor otherwiseundesirable and is acceptable for veterinary as well as humanpharmaceutical use.

The terms “pharmaceutically acceptable excipient”, “pharmaceuticallyacceptable carrier” and “therapeutically inert excipient” can be usedinterchangeably and denote any pharmaceutically acceptable ingredient ina pharmaceutical composition having no therapeutic activity and beingnon-toxic to the subject administered, such as disintegrators, binders,fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants,surfactants, carriers, diluents or lubricants used in formulatingpharmaceutical products.

The terms “individual” or “subject” refer to a mammal. Mammals include,but are not limited to, domesticated animals (e.g., cows, sheep, cats,dogs, and horses), primates (e.g., humans and non-human primates such asmonkeys), rabbits, and rodents (e.g., mice and rats). In certainembodiments, the individual or subject is a human.

The term “therapeutically effective amount” denotes an amount of acompound or molecule of the present invention that, when administered toa subject, (i) treats or prevents the particular disease, condition ordisorder, (ii) attenuates, ameliorates or eliminates one or moresymptoms of the particular disease, condition, or disorder, or (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition or disorder described herein. The therapeuticallyeffective amount will vary depending on the compound, the disease statebeing treated, the severity of the disease treated, the age and relativehealth of the subject, the route and form of administration, thejudgement of the attending medical or veterinary practitioner, and otherfactors.

The terms “treating” or “treatment” of a disease state includeinhibiting the disease state, i.e., arresting the development of thedisease state or its clinical symptoms, or relieving the disease state,i.e., causing temporary or permanent regression of the disease state orits clinical symptoms.

The term “spinal muscular atrophy” (or SMA) relates to a disease causedby an inactivating mutation or deletion in the SMN1 gene on bothchromosomes, resulting in a loss of SMN1 gene function.

Symptoms of SMA include muscle weakness, poor muscle tone, weak cry,weak cough, limpness or a tendency to flop, difficulty sucking orswallowing, difficulty breathing, accumulation of secretions in thelungs or throat, clenched fists with sweaty hand, flickering/vibratingof the tongue, head often tilted to one side, even when lying down, legsthat tend to be weaker than the arms, legs frequently assuming a “froglegs” position, feeding difficulties, increased susceptibility torespiratory tract infections, bowel/bladder weakness, lower-than-normalweight, inability to sit without support, failure to walk, failure tocrawl, and hypotonia, areflexia, and multiple congenital contractures(arthrogryposis) associated with loss of anterior home cells.

The term “treating spinal muscular atrophy (SMA)” or “treatment ofspinal muscular atrophy (SMA)” includes one or more of the followingeffects: (i) reduction or amelioration of the severity of SMA; (ii)delay of the onset of SMA; (iii) inhibition of the progression of SMA;(iv) reduction of hospitalization of a subject; (v) reduction ofhospitalization length for a subject; (vi) increase of the survival of asubject; (vii) improvement of the quality of life of a subject; (viii)reduction of the number of symptoms associated with SMA; (ix) reductionof or amelioration of the severity of one or more symptoms associatedwith SMA; (x) reduction of the duration of a symptom associated withSMA; (xi) prevention of the recurrence of a symptom associated with SMA;(xii) inhibition of the development or onset of a symptom of SMA; and/or(xiii) inhibition of the progression of a symptom associated with SMA.

More particular, the term “treating SMA” denotes one or more of thefollowing beneficial effects: (i) a reduction in the loss of musclestrength; (ii) an increase in muscle strength; (iii) a reduction inmuscle atrophy; (iv) a reduction in the loss of motor function; (v) anincrease in motor neurons; (vii) a reduction in the loss of motorneurons; (viii) protection of SMN deficient motor neurons fromdegeneration; (ix) an increase in motor function; (x) an increase inpulmonary function; and/or (xi) a reduction in the loss of pulmonaryfunction. In further detail, the term “treating SMA” refers to thefunctional ability or retention of the functional ability for a humaninfant or a human toddler to sit up unaided or for a human infant, ahuman toddler, a human child or a human adult to stand up unaided, towalk unaided, to run unaided, to breathe unaided, to turn during sleepunaided, or to swallow unaided.

The term “EC_(1.5×) concentration for production of full length SMN2minigene mRNA” (or “EC_(1.5×) minigene”) is defined as thatconcentration of test compound that is effective in increasing theamount of full length SMN2 minigene mRNA to a level 1.5-fold greaterrelative to that in vehicle-treated cells.

The term “EC_(1.5×) concentration for SMN protein expression” (or“EC_(1.5×) SMN protein”) is defined as that concentration of testcompound that is effective in producing 1.5 times the amount of SMNprotein in an SMA patient fibroblast cell compared to the amountproduced from the vehicle control.

The term “half maximal effective concentration” (EC50) denotes theplasma concentration of a particular compound or molecule required forobtaining 50% of the maximum of a particular effect in vivo.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer or acidifier, excipient, stabilizer, orpreservative.

The term “buffer” or “buffer system” denotes a pharmaceuticallyacceptable excipient or excipient mixture, which stabilizes the pH of apharmaceutical preparation. Suitable buffers are well known in the artand can be found in the literature. Particular pharmaceuticallyacceptable buffers comprise citric buffer, malate buffer, maleatebuffer, or tartrate buffer, most particularly tartrate buffer.Particular buffer systems of the invention combinations of organic acidand selected salts thereof, e.g. tribasic sodium citrate and citricacid, malic acid and sodium malate, potassium sodium tartrate andtartaric acid, or disodium tartrate and tartaric acid, particularlypotassium sodium tartrate and tartaric acid. Alternatively, the organicacid (particularly tartaric acid) can be employed alone as “acidifier”instead of the combination of acid and the corresponding salt.Independently from the buffer used, the pH can be adjusted with an acidor a base known in the art, e.g. hydrochloric acid, acetic acid,phosphoric acid, sulfuric acid and citric acid, sodium hydroxide andpotassium hydroxide. Particular acidifier is tartaric acid.

The term “antioxidant” denotes pharmaceutically acceptable excipients,which prevent oxidation of the active pharmaceutical ingredient.Antioxidants comprise ascorbic acid, glutathione, cysteine, methionine,citric acid, EDTA.

The term “surfactant” denotes a pharmaceutically acceptable excipientwhich is used to protect protein compositions against mechanicalstresses like agitation and shearing. Examples of pharmaceuticallyacceptable surfactants include poloxamers, polysorbates, polyoxyethylenealkyl ethers (BRIJ®), alkylphenylpolyoxyethylene ethers (TRITON-X®) orsodium dodecyl sulfate (SDS).

The term “poloxamer” denotes non-ionic triblock copolymers composed of acentral hydrophobic chain of poly(propylene oxide) (PPO) flanked by twohydrophilic chains of poly(ethylene oxide) (PEO), each PPO or PEO chaincan be of different molecular weights.

Poloxamers are also known by the trade name Pluronics. ParticularPoloxamer is Poloxamer 188, a poloxamer wherein the PPO chain has amolecular mass of 1800 g/mol and a PEO content of 80% (w/w).

The term “polysorbate” denotes oleate esters of sorbitol and itsanhydrides, typically copolymerized with ethylene oxide. Particularpolysorbates are Polysorbate 20 (poly(ethylene oxide) (20) sorbitanmonolaurate, TWEEN 20®) or Polysorbate 80 (poly(ethylene oxide) (80)sorbitan monolaurate, TWEEN 80®).

The “hydrophilic-lipophilic balance” (HLB) value denotes the degree ofhydrophilicity of a non-ionic surfactant. The HLB value is determined bythe ratio between the molecular mass of the hydrophilic portion of thesurfactant molecule and its overall molecular mass, as described byGriffin W. C., Journal of the Society of Cosmetic Chemists (1949) 1:311.

The term “hydrophilic” denotes the capacity of a molecule or portion ofa molecule to interact with polar solvents, in particular with water, orwith other polar moieties driven by hydrogen bonding, dipole-ioninteractions and/or dipole-dipole interactions.

The terms “lipophilic” and “hydrophobic” can be used interchangeably anddenote the tendency of a molecule or portion of a molecule to dissolvein non-polar environment such as fats, oils, and non-polar solventsdriven by London dispersion forces.

The “log P” value denotes the decimal logarithm of the partitioncoefficient P and is a measure of lipophilicity of a neutral unchargedcompound. In instant application, the partition coefficient P isdetermined by the ratio between the concentration of solute in organicphase, particularly 1-octanol, and its concentration in aqueous phase atequilibrium.

Compounds of Formula (I)

In detail, the present invention relates to a pharmaceutical compositioncomprising a compound of formula (I)

-   -   wherein    -   R¹ is hydrogen or C₁₋₇-alkyl;    -   R² is hydrogen, cyano, C₁₋₇-haloalkyl or C₃₋₈-cycloalkyl;    -   R³ is hydrogen, C₁₋₇-alkyl, or C₃₋₈-cycloalkyl;    -   A is N-heterocycloalkyl or NR¹²R¹³, wherein N-heterocycloalkyl        comprises 1 or 2 nitrogen ring atoms and is optionally        substituted with 1, 2, 3 or 4 substituents selected from R¹⁴;    -   R¹² is heterocycloalkyl comprising 1 nitrogen ring atom, wherein        heterocycloalkyl is optionally substituted with 1, 2, 3 or 4        substituents selected from R¹⁴;    -   R¹³ is hydrogen, C₁₋₇-alkyl or C₃₋₈-cycloalkyl;    -   R¹⁴ is independently selected from hydrogen, C₁₋₇-alkyl, amino,        amino-C₁₋₇-alkyl, C₃₋₈-cycloalkyl and heterocycloalkyl or two        R¹⁴ together form C₁₋₇-alkylene;    -   with the proviso that if A is N-heterocycloalkyl comprising only        1 nitrogen ring atom, then at least one R¹⁴ substituent is amino        or amino-C₁₋₇-alkyl;    -   or pharmaceutically acceptable salts thereof;    -   wherein the composition is an oral aqueous solution or a dry        powder suitable for constitution of an oral aqueous solution.

A particular embodiment of the present invention is a pharmaceuticalcomposition comprising a compound of formula (I) or pharmaceuticallyacceptable salts thereof.

Further, it is to be understood that every embodiment relating to aspecific A, R¹, R² or R³ as disclosed herein may be combined with anyother embodiment relating to another A, R¹, R² or R³ as disclosedherein.

In a particular embodiment of the present invention:

-   -   R¹ is hydrogen or C₁₋₇-alkyl;    -   R² is hydrogen, cyano, C₁₋₇-haloalkyl or C₃₋₈-cycloalkyl;    -   R³ is hydrogen, C₁₋₇-alkyl, or C₃₋₈-cycloalkyl;    -   A is N-heterocycloalkyl comprising 1 or 2 nitrogen ring atoms,        wherein N-heterocycloalkyl is optionally substituted with 1, 2,        3 or 4 substituents selected from R¹⁴;

R¹⁴ is independently selected from hydrogen, C₁₋₇-alkyl, amino,amino-C₁₋₇-alkyl, C₃₋₈-cycloalkyl and heterocycloalkyl or two R¹⁴together form C₁₋₇-alkylene;

with the proviso that if A is N-heterocycloalkyl comprising only 1nitrogen ring atom, then at least one R¹⁴ substituent is amino oramino-C₁₋₇-alkyl.

In a particular embodiment of the present invention R¹ is C₁₋₇-alkyl,particularly methyl.

In a particular embodiment of the present invention R² is hydrogen orC₁₋₇-alkyl, particularly hydrogen or methyl.

In a particular embodiment of the present invention R³ is hydrogen orC₁₋₇-alkyl, particularly hydrogen or methyl.

In a particular embodiment of the present invention R¹² is piperidinyloptionally substituted with 1, 2, 3 or 4 substituents selected from R¹⁴.

In a particular embodiment of the present invention R¹³ is hydrogen orC₁₋₇-alkyl, particularly hydrogen or methyl.

In a particular embodiment of the present invention R¹⁴ is independentlyselected from C₁₋₇-alkyl and heterocycloalkyl or two R¹⁴ together formC₁₋₇-alkylene.

In a particular embodiment of the present invention R¹⁴ is independentlyselected from methyl, ethyl and pyrrolidinyl or two R¹⁴ together formethylene.

In a particular embodiment of the present invention A is a saturatedmono- or bicyclic N-heterocycloalkyl comprising 1 or 2 nitrogen atomsand is optionally substituted with 1, 2, 3 or 4 substituents selectedfrom R¹⁴.

In a particular embodiment of the present invention theN-heterocycloalkyl in A or the heterocycloalkyl in R¹² as defined hereinare substituted with 1 or 2 substituents selected from R¹⁴.

In a particular embodiment of the present invention theN-heterocycloalkyl in A as defined herein is further characterized inthat one ring nitrogen atoms is basic.

In a particular embodiment of the present invention A is

-   -   wherein    -   X is N or CH;    -   R⁴ is hydrogen, C₁₋₇-alkyl or —(CH₂)_(m)—NR⁹R¹⁰;    -   R⁵ is hydrogen or C₁₋₇-alkyl;    -   R⁶ is hydrogen or C₁₋₇-alkyl;    -   R⁷ is hydrogen or C₁₋₇-alkyl;    -   R⁸ is hydrogen or C₁₋₇-alkyl;    -   R⁹ and R¹⁰ are independently selected from hydrogen, C₁₋₇-alkyl        and C₃₋₈-cycloalkyl;

R¹³ is hydrogen, C₁₋₇-alkyl or C₃₋₈-cycloalkyl;

-   -   n is 0, 1 or 2;    -   m is 0, 1, 2 or 3;    -   or R⁴ and R⁵ together form a C₁₋₇-alkylene;    -   or R⁴ and R⁷ together form a C₁₋₇-alkylene;    -   or R⁵ and R⁶ together form a C₂₋₇-alkylene;    -   or R⁵ and R⁷ together form a C₁₋₇-alkylene;    -   or R⁵ and R⁹ together form a C₁₋₇-alkylene;    -   or R⁷ and R⁸ together form a C₂₋₇-alkylene;    -   or R⁷ and R⁹ together form a C₁₋₇-alkylene;    -   or R⁹ and R¹⁰ together form a C₂₋₇-alkylene;    -   with the proviso that if X is CH then R⁴ is —(CH₂)_(m)—NR⁹R¹⁰;        and    -   with the proviso that if X is N and R⁴ is —(CH₂)_(m)—NR⁹R¹⁰ then        m is 2 or 3.

It has been found that brain penetration is improved when at least oneof R⁴, R⁵, R⁶, R⁷ and R⁸ is not hydrogen.

In a particular embodiment of the invention at least one of R⁴, R⁵, R⁶,R⁷ and R⁸ is other than hydrogen.

In a particular embodiment of the present invention X is N.

In a particular embodiment of the present invention n is 1.

In a particular embodiment of the present invention R⁴ is hydrogen,methyl or —(CH₂)_(m)—NR⁹R¹⁰, more particularly hydrogen.

In a particular embodiment of the present invention R⁵ is hydrogen,methyl or ethyl, more particularly methyl.

In a particular embodiment of the present invention R⁶ is hydrogen ormethyl, more particularly hydrogen.

In a particular embodiment of the present invention R⁷ is hydrogen ormethyl.

In a particular embodiment of the present invention R⁸ is hydrogen.

In a particular embodiment of the present invention m is 0.

In a particular embodiment of the present invention R⁴ and R⁵ togetherform propylene.

In a particular embodiment of the present invention R⁵ and R⁶ togetherform ethylene;

In a particular embodiment of the present invention R⁹ and R¹⁰ togetherform butylene.

In a particular embodiment of the present invention A is selected fromthe group of:

wherein R⁴, R⁵, R⁶, R⁷, R⁸ and R¹³ are as defined herein and wherein R¹¹is hydrogen or C₁₋₇-alkyl.

In a particular embodiment of the present invention A is selected fromthe group of piperazinyl, diazepanyl, pyrrolidinyl andhexahydropyrrolo[1,2-a]pyrazinyl, each optionally substituted with 1, 2,3 or 4 substituents selected from R¹⁴ as defined herein.

In a particular embodiment of the present invention A is selected fromthe group of piperazin-1-yl, 1,4-diazepan-1-yl, pyrrolidin-1-yl andhexahydropyrrolo[1,2-a]pyrazin-2 (1H)-yl, each optionally substitutedwith 1 or 2 substituents selected from R¹⁴ as defined herein.

In a particular embodiment of the present invention A is NR¹²R¹³,wherein R¹² and R¹³ are as described herein.

In a particular embodiment of the present invention A is selected fromthe group of:

In a particular embodiment of the present invention R¹ is methyl, R² ishydrogen or methyl, R³ is hydrogen, and A is

In a particular embodiment of the present invention R¹ is methyl, R² ismethyl, R³ is hydrogen, and A is

In a particular embodiment of the present invention the compound offormula (I) is selected from the group consisting of:

-   2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3    S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3    S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   7-(1,4-diazepan-1-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3    S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   7-(1,4-diazepan-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(3R,5    S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-(3,3-dimethylpiperazin-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;-   7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-(3,3-dimethylpiperazin-1-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(3R)-3-ethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;    and pharmaceutically acceptable salts thereof.

In a particular embodiment of the present invention the compound offormula (I) is selected from the group consisting of:

-   7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;-   7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;-   7-[(3R,5    S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;-   7-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;    and pharmaceutically acceptable salts thereof.

A particular compound of formula (I) of the present invention is7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneor pharmaceutically acceptable salts thereof.

A particular embodiment of the present invention relates to apharmaceutical composition comprising7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneor a pharmaceutically acceptable salt thereof.

A particular compound of formula (I) of the present invention is7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneor pharmaceutically acceptable salts thereof.

A particular embodiment of the present invention relates to apharmaceutical composition comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneor a pharmaceutically acceptable salt thereof.

Manufacturing Processes

Compounds of formula (I) and pharmaceutically acceptable salts thereofas defined above can be prepared following standard methods known in theart.

As illustrated in Scheme 1, the commercially available amino-pyridine offormula (II) can be reacted with a malonic ester to afford theintermediate of formula (III), wherein Y and R³ are as described hereinand R is C₁₋₂-alkyl, particularly methyl. The compound of formula (III)is then treated with a chlorinating reagent (such as POCl₃ and the like)to provide a compound of formula (IV). The compound of formula (IV) isthen reacted in a Suzuki cross-coupling reaction with a compound offormula (V), wherein R¹ and R² are as described herein and Z is B(OH)₂or an C₁₋₇-alkyl boronic acid ester such as4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl, in the presence of acatalyst (such as (1,1′-bis(diphenylphosphino)ferrocene)palladium(II)dichloride (Pd(dppf)Cl₂) and the like) and a base (such as K₂CO₃ and thelike) in a suitable solvent (such as DMF and the like), to afford thecompound of formula (VI). Finally, the compound of formula (VI) isreacted with a compound M-A either in:

-   -   a) an aromatic nucleophilic substitution reaction (particularly        if Y is fluoro) by heating at a temperature from 80° C. to 200°        C.; or    -   b) a Buchwald-Hartwig amination reaction in the presence of a        palladium catalyst (e.g. tetrakis(triphenylphosphine)palladium        (Pd(PPh₃)₄) or bis(dibenzylideneacetone)palladium (Pd(dba)₂) by        heating at a temperature from 20° C. to 100° C.;

in a solvent (e.g. dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP),or dimethylformamide (DMF)) to give a compound of formula (I), wherein Ais as defined herein, M is hydrogen, sodium or potassium, particularlyhydrogen, and wherein M is linked to A via a nitrogen atom of A.

In one embodiment, the invention relates to a process for themanufacture of compounds of formula (I) and pharmaceutically acceptablesalts thereof as defined above, comprising the reaction of a compound offormula (VI) with a compound M-A either in:

-   -   a) an aromatic nucleophilic substitution reaction (particularly        if Y is fluoro) by heating at a temperature from 80° C. to 200°        C.; or    -   b) a Buchwald-Hartwig amination reaction in the presence of a        palladium catalyst (e.g. tetrakis(triphenylphosphine)palladium        (Pd(PPh₃)₄) or bis(dibenzylideneacetone)palladium Pd(dba)₂) by        heating at a temperature from 20° C. to 100° C.;

in a solvent (e.g. dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP),or dimethylformamide (DMF)), wherein A, Y, R¹, R² and R³ are as definedherein, M is hydrogen, sodium or potassium, particularly hydrogen, andwherein M is linked to A via a nitrogen atom of A.

A particular embodiment of the invention relates to a process for thepreparation of compounds of formula (I) and pharmaceutically acceptablesalts thereof as defined above, comprising an aromatic nucleophilicsubstitution reaction between a compound of formula (VI) as describedabove with a compound of formula M-A by heating in a solvent, wherein A,R¹, R², R³ and Y are as defined above, M is hydrogen, sodium orpotassium, and wherein M is linked to A via a nitrogen atom of A.

A particular embodiment of the invention relates to a process for thepreparation of compounds of formula (I) and pharmaceutically acceptablesalts thereof as defined above, wherein the aromatic nucleophilicsubstitution reaction is performed at a temperature from 80° C. to 200°C.

A particular embodiment of the invention relates to a process for thepreparation of compounds of formula (I) and pharmaceutically acceptablesalts thereof as defined above, wherein the solvent of the aromaticnucleophilic substitution reaction is selected from dimethyl sulfoxide(DMSO), N-methylpyrrolidone (NMP), and dimethylformamide (DMF).

A particular embodiment of the invention relates to a process for thepreparation of compounds of formula (I) and pharmaceutically acceptablesalts thereof as defined above, wherein M is hydrogen.

Particularly, compounds of formula (I) and pharmaceutically acceptablesalts thereof can be prepared in accordance to the methods described inthe examples herein.

Medical Uses

As described above, the compounds of formula (I) and theirpharmaceutically acceptable salts possess valuable pharmacologicalproperties and have been found to enhance inclusion of exon 7 of SMN1and/or SMN2 into mRNA transcribed from the SMN1 and/or SMN2 gene,thereby increasing expression of SMN protein in a human subject in needthereof.

The compounds of the present invention can be used, either alone or incombination with other drugs, for the treatment, prevention, delayingprogression and/or amelioration of diseases caused by an inactivatingmutation or deletion in the SMN1 gene and/or associated with loss ordefect of SMN1 gene function. These diseases include, but are notlimited to spinal muscular atrophy (SMA).

A particular embodiment of the present invention relates to apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof as defined above and one ormore pharmaceutically acceptable excipients for the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, particularly forthe treatment, prevention, delaying progression and/or amelioration ofSMA.

A particular embodiment of the present invention relates to apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof as defined above for use astherapeutically active substances, especially for use as therapeuticallyactive substances for the treatment, prevention, delaying progressionand/or amelioration of diseases caused by an inactivating mutation ordeletion in the SMN1 gene and/or associated with loss or defect of SMN1gene function, particularly for the treatment, prevention, delayingprogression and/or amelioration of spinal muscular atrophy (SMA).

A particular embodiment of the present invention relates to apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof as defined above for the use inthe treatment, prevention, delaying progression and/or amelioration ofdiseases caused by an inactivating mutation or deletion in the SMN1 geneand/or associated with loss or defect of SMN1 gene function,particularly for use in the treatment, prevention, delaying progressionand/or amelioration of spinal muscular atrophy (SMA).

A particular embodiment of the present invention relates to a method forthe treatment, prevention, delaying progression and/or amelioration ofdiseases caused by an inactivating mutation or deletion in the SMN1 geneand/or associated with loss or defect of SMN1 gene function,particularly for the treatment, prevention, delaying progression and/oramelioration of spinal muscular atrophy (SMA), which method comprisesadministering a pharmaceutical composition comprising a compound offormula (I) or a pharmaceutically acceptable salt thereof as definedabove to a subject.

A particular embodiment of the present invention relates to the use of apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof as defined above for thetreatment, prevention, delaying progression and/or amelioration ofdiseases caused by an inactivating mutation or deletion in the SMN1 geneand/or associated with loss or defect of SMN1 gene, particularly for thetreatment, prevention, delaying progression and/or amelioration ofspinal muscular atrophy (SMA).

A particular embodiment of the present invention relates to the use of apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof as defined above for thepreparation of medicaments for the treatment, prevention, delayingprogression and/or amelioration of diseases caused by an inactivatingmutation or deletion in the SMN1 gene and/or associated with loss ordefect of SMN1 gene function, particularly for the treatment,prevention, delaying progression and/or amelioration of spinal muscularatrophy (SMA). Such medicaments comprise compounds of formula (I) ortheir pharmaceutically acceptable salts as defined above.

Combinations

Cytoprotectors (such as olesoxime) and SMN2 gene splicing modulators(such as the compounds of formula (I)) are complementary approaches totreating Spinal Muscular Atrophy (SMA). There is supportive evidence tosuggest that co-administration of cytoprotectors and SMN2 gene splicingmodulators as a combination treatment will provide additional benefit toall types of SMA patients. The extent of added benefit can be quantifiedthrough studies of combination treatment in SMA mouse models.

A particular SMN2 gene splicing modulator of the invention is a compoundof formula (I) as described herein or a pharmaceutically acceptable saltthereof.

A particular cytoprotector of the invention is olesoxime.

Systemically low levels of SMN protein cause SMA. α-Motor neurons of thespinal cord are considered particularly vulnerable in this geneticdisorder and their dysfunction and loss cause progressive muscleweakness, paralysis and eventually premature death of afflictedindividuals. Historically, SMA was therefore considered a motorneuron-autonomous disease. However, depletion of SMN in motor neurons ofnormal mice elicited only a very mild phenotype (Park et al, J Neurosci.2010 Sep. 8; 30(36):12005-19). Conversely, restoration of SMN to motorneurons in an SMA mouse model had only modest effects on the SMAphenotype and survival (Hua et al Nature. 2011 Oct. 5; 478(7367):123-6).Collectively, these results suggest that additional cell typescontribute to the pathogenesis of SMA, and understanding thenon-autonomous requirements is crucial for developing effectivetherapies.

Current research points to SMA as a multi-cell, -tissue, -systemdisorder. There are several in vitro, in vivo studies, as well as humancase studies showing that a variety of tissues are affected in SMA, suchas: afferent nerves, muscle, vasculature, brain, heart and pancreas(Hamilton and Gillingwater, et al, Trends Mol Med. 2013 January;19(1):40-50. For example, there is a body of work that shows a muscleintrinsic defects in SMA, indicating that SMN plays a role in muscledevelopment and regeneration (Boyer et al, Front Physiol. 2013 Dec. 18;4:356. Hayhurst M et al., Dev Biol. 2012 Aug. 15; 368(2):323-34;Briccino et al, Hum Mol Genet. 2014 Sep. 15; 23(18):4745-57. Shafey etal, Exp Cell Res. 2005 Nov. 15; 311(1):49-61., Cifuentes-Diaz et al, JCell Biol. 2001 Mar. 5; 152(5):1107-14. Martinez et al J Neurosci. 2012Jun. 20; 32(25):8703-15. This highlights the importance ofmuscle-targeted treatments for SMA patients. Many therapeutic strategiestarget restoring SMN protein. Antisense oligonucleotides and genetherapy potential treatments target increasing SMN in the CNS tissuealone. Effectively targeting treatment to other key affected cells andtissues remains a challenge.

The cytoprotector olesoxime and the SMN2 gene splicing modulators offormula (I) can both be administered orally and are distributedsystemically. Moreover, they have complementary mechanisms of action.Olesoxime is cytoprotective by preserving the mitochondrial membrane andpreventing mitochondrial dysfunction, an important element of diseasepathophysiology in SMA. Mitochondria are particularly abundant inenergy-demanding cells, such as motor neurons and muscle cells, bothidentified as target treatment tissues in SMA. The SMN2 gene splicingmodulators of formula (I) target increasing SMN protein systemically.

The SMN2 gene splicing modulators of formula (I) correct SMN protein atthe RNA level through correcting mis-splicing of the SMN pre-mRNA. Themaximal increase in SMN protein in SMA motor neurons and fibroblastsabove untreated cells resulted in a similar increase in both cell types(60-80%). Moreover, in both the severe and mild models of SMA, micetreated with SMN2 gene splicing modulators of formula (I) had anincrease in SMN protein reaching approximately 43% (brain) and 55%(muscle) of protein levels in heterozygous mice. The increase in proteinwas sufficient to provide substantial benefit, restoring connectivity atthe neuromuscular junctions (NMJ), and on survival in the severe micetreated with compounds of formula (I). Given that the increase inprotein is not corrected to 100% of heterozygous mice or wildtype mice,it is reasonable to believe that there may be additional improvementswith co-treatments, especially treatments that target other mechanismsof disease pathogenesis.

In vitro binding studies and oxidative stress assays indicate that thecytoprotector olesoxime preserves the mitochondrial membrane in adisease where there is evidence of mitochondrial dysfunction (SMA),thereby preventing cell death. In vitro fluorescence imaging experimentsshowed that olesoxime accumulates at the membrane level of mitochondriain neurons. In addition, in olesoxime treated SOD1 mice (severe familialALS model) the neuromuscular junctions (NMJ) were preserved when treatedearly. Thus, olesoxime could provide additional and complementarybenefit through mitochondrial and cytoprotective mechanisms.

The additional benefit of the combination treatment of the cytoprotectorolesoxime and the SMN2 gene splicing modulators of formula (I) are beingconfirmed in a mild mouse model of SMA. The mild model was generated bytreating the severe SMA delta7 mouse model with a low dose of an SMNsplicing modulator. In the study, the cytoprotector olesoxime and theSMN2 gene splicing modulators of formula (I) are tested alone and incombination. The impact of treatment on mouse NMJs is the primaryendpoint of these studies, given the importance of this phenotype.Oxidative stress markers in muscle tissues are also evaluated.

A compound of formula (I) or a pharmaceutically acceptable salt thereofcan be combined with olesoxime in one single pharmaceutical composition(e.g. a fixed dose combination) or a compound of formula (I) or apharmaceutically acceptable salt thereof and olesoxime can beco-administered sequentially one after the other.

As described herein, the co-administration of a compound of formula (I)and olesoxime can have beneficial and synergistic effects for thetreatment, prevention, delaying progression and/or amelioration ofdiseases caused by an inactivating mutation or deletion in the SMN1 geneand/or associated with loss or defect of SMN1 gene function, andadditionally for the protection of cells implicated in thepathophysiology of the disease, particularly for the treatment,prevention, delaying progression and/or amelioration of spinal muscularatrophy (SMA).

In the context of the present invention, the term “co-administration” oftwo API can be simultaneous, almost simultaneous, or delayed in time bya few days or weeks, for example by up to 4 or 5 weeks.

The compounds of the present invention can be used in combination withcytoprotectors such as olesoxime for the treatment, prevention, delayingprogression and/or amelioration of diseases caused by an inactivatingmutation or deletion in the SMN1 gene and/or associated with loss ordefect of SMN1 gene function, and additionally for the protection ofcells implicated in the pathophysiology of the disease. These diseasesinclude, but are not limited to spinal muscular atrophy (SMA).

In a particular embodiment, the present invention would show a synergyof a combination of a compound of formula (I) with olesoxime eitheralone or in combination thereof in the treatment, prevention, delayingprogression and/or amelioration of diseases caused by an inactivatingmutation or deletion in the SMN1 gene and/or associated with loss ordefect of SMN1 gene function, and additionally for the protection ofcells implicated in the pathophysiology of the disease, particularly forthe treatment, prevention, delaying progression and/or amelioration ofspinal muscular atrophy (SMA).

A particular embodiment of the present invention relates to apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and olesoxime and one or morepharmaceutically acceptable excipients for use in the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, and additionallyfor the protection of cells implicated in the pathophysiology of thedisease, particularly for the treatment, prevention, delayingprogression and/or amelioration of SMA.

A particular embodiment of the present invention relates to apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and olesoxime for use astherapeutically active substances, especially for use as therapeuticallyactive substances for the treatment, prevention, delaying progressionand/or amelioration of diseases caused by an inactivating mutation ordeletion in the SMN1 gene and/or associated with loss or defect of SMN1gene function, and additionally for the protection of cells implicatedin the pathophysiology of the disease, particularly for the treatment,prevention, delaying progression and/or amelioration of spinal muscularatrophy (SMA).

A particular embodiment of the present invention relates to apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and olesoxime for use in thetreatment, prevention, delaying progression and/or amelioration ofdiseases caused by an inactivating mutation or deletion in the SMN1 geneand/or associated with loss or defect of SMN1 gene function andadditionally for the protection of cells implicated in thepathophysiology of the disease, particularly for use in the treatment,prevention, delaying progression and/or amelioration of spinal muscularatrophy (SMA).

A particular embodiment of the present invention relates to a method forthe treatment, prevention, delaying progression and/or amelioration ofdiseases caused by an inactivating mutation or deletion in the SMN1 geneand/or associated with loss or defect of SMN1 gene function andadditionally for the protection of cells implicated in thepathophysiology of the disease, particularly for the treatment,prevention, delaying progression and/or amelioration of spinal muscularatrophy (SMA), which method comprises administering a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof and olesoxime to a subject.

A particular embodiment of the present invention relates to the use of apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and olesoxime for thetreatment, prevention, delaying progression and/or amelioration ofdiseases caused by an inactivating mutation or deletion in the SMN1 geneand/or associated with loss or defect of SMN1 gene function andadditionally for the protection of cells implicated in thepathophysiology of the disease, particularly for the treatment,prevention, delaying progression and/or amelioration of spinal muscularatrophy (SMA).

A particular embodiment of the present invention relates to the use of acompound of formula (I) or a pharmaceutically acceptable salt thereofand olesoxime for the preparation of medicaments for the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, and additionallyfor the protection of cells implicated in the pathophysiology of thedisease, particularly for the treatment, prevention, delayingprogression and/or amelioration of spinal muscular atrophy (SMA). Suchmedicaments comprise compounds of formula (I) or their pharmaceuticallyacceptable salts and olesoxime.

The present invention therefore also encompasses a kit for thepreparation of pharmaceutical compositions comprising a compound offormula (I) or a pharmaceutically acceptable salt thereof and olesoxime.

In one embodiment the invention provides a kit comprising (a) a firstpharmaceutical composition comprising a therapeutically effective amountof (i) a compound of formula (I), and (ii) a pharmaceutically acceptablecarrier, (b) a second pharmaceutical composition comprising (i)olesoxime, and (ii) a pharmaceutically acceptable carrier, (c)prescribing information, and (d) a container, wherein the prescribinginformation includes advice to a patient regarding co-administration ofthe two API.

In another embodiment the invention provides a kit comprising acomposition comprising a therapeutically effective amount of a compoundof formula (I) and olesoxime, prescribing information also known as“leaflet”, a blister package or bottle (HDPE or glass) and a container.

The term “kit” as used herein refers to a collection of theaforementioned components which may be provided separately or within asingle container. The container optionally comprises instructions forcarrying out the method of the present disclosure.

One embodiment of the invention provides a combination of a SMN2 genesplicing modulator and a cytoprotector.

One embodiment of the invention provides a combination of a SMN2 genesplicing modulator and a cytoprotector, for use in the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, and/or for theprotection of cells implicated in the pathophysiology of the disease.

One embodiment of the invention provides a combination of a compound offormula (I) according to any of claims 1 to 9 or a pharmaceuticallyacceptable salt thereof and olesoxime, for use in the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, and/or for theprotection of cells implicated in the pathophysiology of the disease.

One embodiment of the invention provides a method for the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, and/or for theprotection of cells implicated in the pathophysiology of the disease,which method comprises administering a combination of a SMN2 genesplicing modulator and a cytoprotector to a subject.

One embodiment of the invention provides a method for the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, and/or for theprotection of cells implicated in the pathophysiology of the disease,which method comprises administering a combination of a compound offormula (I) according to any of claims 1 to 9 or a pharmaceuticallyacceptable salt thereof and olesoxime to a subject.

One embodiment of the invention provides the use of a combination of aSMN2 gene splicing modulator and a cytoprotector for the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, and/or for theprotection of cells implicated in the pathophysiology of the disease.

One embodiment of the invention provides the use of a combination of acompound of formula (I) according to any of claims 1 to 9 or apharmaceutically acceptable salt thereof and olesoxime for thetreatment, prevention, delaying progression and/or amelioration ofdiseases caused by an inactivating mutation or deletion in the SMN1 geneand/or associated with loss or defect of SMN1 gene function, and/or forthe protection of cells implicated in the pathophysiology of thedisease.

One embodiment of the invention provides the use of a combination of aSMN2 gene splicing modulator and a cytoprotector for the preparation ofmedicaments for the treatment, prevention, delaying progression and/oramelioration of diseases caused by an inactivating mutation or deletionin the SMN1 gene and/or associated with loss or defect of SMN1 genefunction, and/or for the protection of cells implicated in thepathophysiology of the disease.

One embodiment of the invention provides the use of a combination of acompound of formula (I) according to any of claims 1 to 9 or apharmaceutically acceptable salt thereof and olesoxime for thepreparation of medicaments for the treatment, prevention, delayingprogression and/or amelioration of diseases caused by an inactivatingmutation or deletion in the SMN1 gene and/or associated with loss ordefect of SMN1 gene function, and/or for the protection of cellsimplicated in the pathophysiology of the disease.

Pharmaceutical Compositions

It has been found, that the compounds of formula (I) of presentinvention have a high aqueous solubility. Due to the handicaps inswallowing of all age groups of SMA patients, administration of asolution has been found to be preferred.

The compounds of formula (I) can be formulated as oral aqueous solutionby dissolving the drug substance in a buffer system at pH of less thanpH 4, particularly less than pH 3.8, more particularly less than pH 3.6,most particularly pH 3.4, in order to provide sufficiently high drugconcentration, e.g. citric buffer system, malate buffer system, maleatebuffer system, or tartrate buffer system, most particularly tartratebuffer system.

Long term stability of formulations of the compounds of formula (I) canbe achieved by preparing a dry powder or granulation for constitution ofan oral solution. A buffer system can be incorporated into dryformulation by the selection of organic acid and salts thereof as finepowders, e.g. tribasic sodium citrate and citric acid, disodium malateand malic acid, potassium sodium tartrate and tartaric acid, or disodiumtartrate and tartaric acid, particularly potassium sodium tartrate andtartaric acid. Alternatively, the organic acid (particularly tartaricacid) can be employed alone as acidifier instead of the combination ofacid and the corresponding salt.

Powders or granules comprising a compound of formula (I) may comprise adiluent, such as sorbitol, isomalt, or particularly mannitol, andcombinations thereof, which ensure fast dissolution of the powder blendduring constitution of the oral solution. In introduction of a fillerthe powder blend can be granulated by dry compaction in order to improvethe flowability and to ensure robust uniformity.

Ingredients for the constitution of a solvent system for the compoundsof formula (I) can be formulated as separate formulation. Theconstituted solvent can be used for dissolution of the compounds offormula (I) in a bottle at the start of the in-use period of the oralsolution.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof in powder form for constitution of an oralsolution.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof are filled in a multidose bottle with adapterfor use of oral dispensers. It has been found that such multidose bottlewith adapter for use of oral dispensers enables high dosing flexibility,e.g. body weight adjusted dosing and provides safe and convenient doseadministration.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof is prepared through dry granulation by rollercompaction followed bottle filling. It has been found that suchprocessing is beneficial (particularly for water soluble fillers) toprevent demixing.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is an oral aqueoussolution or a dry powder suitable for constitution of an oral aqueoussolution.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is an oral aqueoussolution not including aerosols or a dry powder suitable forconstitution of an oral aqueous solution.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof is not an aerosol.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof does not comprise a tonicifier, e.g. a salt suchas sodium chloride.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is an oral aqueoussolution or a dry powder suitable for constitution of an oral aqueoussolution, and wherein the oral aqueous solution has a pH of less thanpH4, particularly less than pH3.8, more particularly less than pH 3.6,most particularly pH 3.4.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof and a citrate, malate, maleate or tartratebuffer system, particularly a malate or tartrate buffer system, mostparticularly a tartrate buffer system; or alternatively thecorresponding acid of a buffer system alone as acidifier, particularlytartaric acid; wherein the composition is an oral aqueous solution or adry powder suitable for constitution of an oral aqueous solution.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is an oral aqueoussolution.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is an oral aqueoussolution in a buffer system at pH of less than pH4, particularly lessthan pH3.8, more particularly less than pH 3.6, most particularly pH3.4.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is an oral aqueoussolution in a citrate, malate, maleate or tartrate buffer system,particularly in a malate or tartrate buffer system, most particularly ina tartrate buffer system; or alternatively the corresponding acid of abuffer system alone as acidifier, particularly tartaric acid.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is dry powder suitablefor constitution of an oral aqueous solution.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is dry powder comprisinga buffer system suitable for constitution of an oral aqueous solution atpH of less than pH4, particularly less than pH3.8, more particularlyless than pH 3.6, most particularly pH 3.4.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof wherein the composition is dry powder comprisingcitrate, malate, maleate or tartrate buffer system, particularly in amalate or tartrate buffer system, most particularly in a tartrate buffersystem; or alternatively the corresponding acid of a buffer system aloneas acidifier, particularly tartaric acid; suitable for constitution ofan oral aqueous solution.

In one embodiment of the invention, the pharmaceutical compositioncomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof optionally further comprises an extragranular filler, suchas lactose, starch, hydrolyzed starch, maltodextrin, microcrystallinecellulose, mannitol, sorbitol, sucrose, dextrose, dibasic calciumphosphate, calcium sulfate, or combinations thereof.

In a particular embodiment of the invention, the extragranular filler issorbitol, isomalt, mannitol, or combinations thereof, particularlymannitol, more particularly crystalline mannitol, most particularlycrystalline mannitol with mean diameter of 160 μm (Pearlitol® 160C).

In introduction of a diluent, the powder blend can be granulated by drycompaction in order to improve the flowability and to ensure robustuniformity.

In one embodiment of the invention, the pharmaceutical compositioncomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof optionally further comprises a diluent, such as lactose,starch, hydrolyzed starch, maltodextrin, microcrystalline cellulose,mannitol, isomalt (E 953,(2ξ)-6-O-α-D-Glucopyranosyl-D-arabino-hexitol), sorbitol, sucrose,dextrose, dibasic calcium phosphate, calcium sulfate, or combinationsthereof.

In one embodiment of the invention, the pharmaceutical compositioncomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof optionally further comprises a diluent, such as lactose,starch, hydrolyzed starch, microcrystalline cellulose, mannitol,sorbitol, sucrose, dextrose, dibasic calcium phosphate, calcium sulfate,or combinations thereof.

In a particular embodiment of the invention, the diluent is mannitol,particularly D-mannitol suitable for direct compression such as Parteck®M100.

In a particular embodiment of the invention, the diluent is a mixture ofmannitol and isomalt, particularly D-mannitol and(2ξ)-6-O-α-D-Glucopyranosyl-D-arabino-hexitol).

Isomalt as second diluent has been found by the inventors of presentinvention to improve the granule properties.

The constituted oral solution of the compounds of formula (I) in abuffer can provide in-use times of more than two weeks by the use ofpreservatives, stabilizers and antioxidants, such as vitamin A, vitaminC, vitamin E, vitamin E TPGS, retinyl palmitate, selenium, cysteine,methionine, citric acid, sodium citrate, methyl paraben, propyl paraben,disodium edetate, butyl hydroxyl toluol, riboflavin, ascorbic acid orcombinations thereof.

The constituted oral solution of the compounds of formula (I) in abuffer can provide in-use times of more than two weeks by the use ofpreservatives, stabilizers and antioxidants, such as vitamin E TPGS,disodium edetate, butyl hydroxyl toluol, riboflavin, ascorbic acid, orcombinations thereof.

In one embodiment of the invention, the pharmaceutical compositioncomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof optionally further comprises a preservative, antioxidantand/or stabilizer, such as vitamin E TPGS (D-alpha tocopherylpolyethylene glycol 1000 succinate), disodiumethylenediaminetetraacetate (disodium edetate, Na₂ EDTA), butyl hydroxyltoluol, riboflavin, ascorbic acid, or combinations thereof. It has beenfound that a preservative, antioxidant and/or stabilizer can bebeneficial for prolonged use time in multidose containers or to improvedrug stability in solution over in-use period.

In a particular embodiment of the invention, the preservative is sorbicacid or sodium benzoate (E211), particularly sodium benzoate.

For pediatric formulations the amount of preservative included should beas low as possible. It has been found that compositions of theinventions with preservative concentrations as low as 1% wt are yieldingstable solutions.

In a particular embodiment of the invention, the antioxidant is ascorbicacid ((5R)-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one).

In a particular embodiment of the invention, the stabilizer is disodiumethylenediaminetetraacetate (disodium edetate, Na₂ EDTA).

In one embodiment of the invention, the pharmaceutical compositioncomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof optionally further comprises a lubricant. It has been foundthat a lubricant can be used as processing aid for roller compaction.Further a lubricant can be used for water soluble ingredients such asPEG to ensure acceptability of appearance.

In a particular embodiment of the invention, the lubricant ispoly(ethylene glycol), particularly poly(ethylene glycol) with numberaverage molecular weight Mn 6,000 (PEG 6000).

In one embodiment of the invention, the pharmaceutical compositioncomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof optionally further comprises a sweetener and/or flavor toimprove palatability.

In a particular embodiment of the invention, the flavor is strawberryflavor or vanilla flavor.

In a particular embodiment of the invention, the sweetener is sucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside,E955) or sodium saccharin.

In a particular embodiment of the invention, the compound of formula (I)is7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof; and    -   a buffer system selected from citrate, malate, maleate or        tartrate, particularly malate or tartrate, most particularly        tartrate; or alternatively the corresponding acid of a buffer        system alone as acidifier, particularly tartaric acid.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof;    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid; and    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof; and    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof;    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   an antioxidant, particularly ascorbic acid; and    -   a stabilizer, particularly disodium edetate.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof;    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid; and    -   a stabilizer, particularly disodium edetate.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof;    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate; and    -   a lubricant, particularly PEG6000.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof;    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   a lubricant, particularly PEG6000; and    -   a preservative, particularly sorbic acid or sodium benzoate,        most particularly sodium benzoate.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof;    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   a lubricant, particularly PEG6000;    -   a preservative, particularly sorbic acid or sodium benzoate,        most particularly sodium benzoate,    -   optionally a sweetener, particularly sucralose or sodium        saccharin, most particularly sucralose; and    -   optionally a flavor, particularly strawberry flavor or vanilla        flavor.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   1 to 10% wt of a compound of formula (I) or a pharmaceutically        acceptable salt thereof;    -   5 to 15% wt of a buffer system, particularly a buffer system        selected from citrate, malate, maleate or tartrate, more        particularly malate or tartrate, most particularly tartrate; or        alternatively the corresponding acid of a buffer system alone as        acidifier, particularly tartaric acid;    -   40 to 70% wt of a diluent, particularly mannitol or a mixture of        mannitol and isomalt, more particularly mannitol;    -   1 to 4% wt of an antioxidant, particularly ascorbic acid;    -   0.5 to 2% wt of a stabilizer, particularly disodium edetate;    -   0.5 to 2% w of a lubricant, particularly PEG6000;    -   1 to 8% wt of a preservative, particularly sorbic acid or sodium        benzoate, most particularly sodium benzoate,    -   0 to 3% wt of a sweetener, particularly sucralose or sodium        saccharin, most particularly sucralose; and    -   0 to 20% wt of a flavor, particularly strawberry flavor or        vanilla flavor;    -   wherein the total amount of ingredients does not exceed 100% wt.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   2 to 6% wt of        7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one        or a pharmaceutically acceptable salt thereof;    -   9 to 13% wt of a tartrate buffer system;    -   45 to 55% wt of a mannitol as first diluent and 8 to 10% wt of        isomalt as second diluent;    -   1 to 3% wt of ascorbic acid as antioxidant;    -   0.5 to 2% wt of disodium edetate as stabilizer;    -   0.5 to 2% w of PEG6000 as lubricant;    -   1 to 7% wt of sodium benzoate as preservative,    -   1.5 to 2% wt of sucralose as sweetener; and    -   13 to 17% wt of strawberry flavor;

wherein the total amount of ingredients does not exceed 100% wt.

Another embodiment of the invention relates to a kit for the preparationof pharmaceutical compositions comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, wherein the kit comprises:

-   -   a powder blend comprising a compound of formula (I) or a        pharmaceutically acceptable salt thereof, and    -   water as solvent for constitution.

Another embodiment of the invention relates to a kit for the preparationof pharmaceutical compositions comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, wherein the kit comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof,    -   a powder blend as vehicle for constitution, and    -   optionally water as solvent for constitution.

Another embodiment relates to a power blend as vehicle suitable forconstitution of a compound of formula (I) as described herein or apharmaceutically acceptable salt thereof, comprising:

-   -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid; and    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol.

Another embodiment relates to a power blend as vehicle suitable forconstitution of a compound of formula (I) as described herein or apharmaceutically acceptable salt thereof, comprising:

-   -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   a lubricant, particularly PEG6000; and    -   a preservative, particularly sorbic acid or sodium benzoate,        most particularly sodium benzoate.

Another embodiment relates to a power blend as vehicle suitable forconstitution of a compound of formula (I) as described herein or apharmaceutically acceptable salt thereof, comprising:

-   -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   a lubricant, particularly PEG6000;    -   a preservative, particularly sorbic acid or sodium benzoate,        most particularly sodium benzoate,    -   optionally a sweetener, particularly sucralose or sodium        saccharin, most particularly sucralose; and    -   optionally a flavor, particularly strawberry flavor or vanilla        flavor.

Another embodiment relates to a power blend as vehicle suitable forconstitution of a compound of formula (I) as described herein or apharmaceutically acceptable salt thereof, comprising:

-   -   4 to 15% wt of a buffer system, particularly a buffer system        selected from citrate, malate, maleate or tartrate, more        particularly malate or tartrate, most particularly tartrate; or        alternatively the corresponding acid of a buffer system alone as        acidifier, particularly tartaric acid;    -   40 to 70% wt of a diluent, particularly mannitol or a mixture of        mannitol and isomalt, more particularly mannitol;    -   1 to 4% wt of an antioxidant, particularly ascorbic acid;    -   0.2 to 2% wt of a stabilizer, particularly disodium edetate;    -   0.5 to 2% w of a lubricant, particularly PEG6000;    -   1 to 8% wt of a preservative, particularly sorbic acid or sodium        benzoate, most particularly sodium benzoate,    -   0 to 3% wt of a sweetener, particularly sucralose or sodium        saccharin, most particularly sucralose; and    -   0 to 20% wt of a flavor, particularly strawberry flavor or        vanilla flavor;

wherein the total amount of ingredients does not exceed 100% wt.

Another embodiment relates to a power blend as vehicle suitable forconstitution of a compound of formula (I) as described herein or apharmaceutically acceptable salt thereof, comprising:

-   -   9 to 13% wt of a tartrate buffer system or tartaric acid;    -   45 to 55% wt of a mannitol as first diluent and 8 to 10% wt of        isomalt as second diluent;    -   1 to 3% wt of ascorbic acid as antioxidant;    -   0.3 to 0.9% wt of disodium edetate as stabilizer;    -   0.5 to 2% w of PEG6000 as lubricant;    -   3 to 7% wt of sodium benzoate as preservative,    -   0.8 to 2.0% wt of sucralose as sweetener; and    -   7.5 to 19% wt of strawberry flavor;

wherein the total amount of ingredients does not exceed 100% wt.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof as described herein and further olesoxime.

In a particular embodiment of the invention olesoxime has a particlesize distribution with d90 value smaller 200 μm, particularly d90 valuesmaller 100 μm, more particularly d90 value of 50-100 μm.

The term “d90 value” denotes the diameter where 90 wt % of the particlesof the ensemble have a smaller equivalent spherical diameter than thevalue.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof as described herein, olesoxime and an oilselected from sesame oil, olive oil, soya oil, cotton oil, castor oil,nut oil, rapeseed oil, corn oil, almond oil, sunflower oil, andcombinations thereof.

A particular embodiment of the invention relates to a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof as described herein; olesoxime; an oil selectedfrom sesame oil, olive oil, soya oil, cotton oil, castor oil, nut oil,rapeseed oil, corn oil, almond oil, sunflower oil, and combinationsthereof; an emulsifying and/or lipophilic solubilizing agents selectedfrom glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul GMO™), glycerylmono-linoleate (Maisine 35-1™), sorbitan mono-oleate (Span 80™), oleicacid, and combinations thereof; and optionally a polar surfactant,particularly a surfactant with a HLB value of less than 7, moreparticularly polysorbate 80 (Tween 80™), caprylocaproyl polyoxylglycerides (Labrasol™), and combinations thereof.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof;    -   a buffer system selected from citrate, malate, maleate or        tartrate, particularly malate or tartrate, most particularly        tartrate; or alternatively the corresponding acid of a buffer        system alone as acidifier, particularly tartaric acid;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof; and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   olesoxime,    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   a lubricant, particularly PEG6000;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   a lubricant, particularly PEG6000;    -   a preservative, particularly sorbic acid or sodium benzoate,        most particularly sodium benzoate;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

In a particular embodiment of the invention, the pharmaceuticalcomposition comprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof    -   a buffer system, particularly a buffer system selected from        citrate, malate, maleate or tartrate, more particularly malate        or tartrate, most particularly tartrate; or alternatively the        corresponding acid of a buffer system alone as acidifier,        particularly tartaric acid;    -   a diluent, particularly mannitol or a mixture of mannitol and        isomalt, more particularly mannitol;    -   an antioxidant, particularly ascorbic acid;    -   a stabilizer, particularly disodium edetate;    -   a lubricant, particularly PEG6000;    -   a preservative, particularly sorbic acid or sodium benzoate,        most particularly sodium benzoate;    -   optionally a sweetener, particularly sucralose or sodium        saccharin, most particularly sucralose;    -   optionally a flavor, particularly strawberry flavor or vanilla        flavor;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

Another embodiment of the invention relates to a kit for the preparationof pharmaceutical compositions comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and olexosime, wherein the kitcomprises:

-   -   a powder blend comprising a compound of formula (I) or a        pharmaceutically acceptable salt thereof;    -   water as solvent for constitution;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

Another embodiment of the invention relates to a kit for the preparationof pharmaceutical compositions comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and olesoxime, wherein the kitcomprises:

-   -   a compound of formula (I) or a pharmaceutically acceptable salt        thereof    -   a powder blend as vehicle for constitution;    -   optionally water as solvent for constitution;    -   olesoxime;    -   an oil, particularly sesame oil, olive oil, soya oil, cotton        oil, castor oil, nut oil, rapeseed oil, corn oil, almond oil,        sunflower oil, or combinations thereof, most particularly sesame        oil;    -   an emulsifying and/or lipophilic solubilizing agents,        particularly glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul        GMO™), glyceryl mono-linoleate (Maisine 35-1™), sorbitan        mono-oleate (Span 80™), oleic acid, or combinations thereof and    -   optionally a polar surfactant, particularly a surfactant with a        HLB value of less than 7, more particularly polysorbate 80        (Tween 80™), caprylocaproyl polyoxyl glycerides (Labrasol™), or        combinations thereof.

FIGURES

FIG. 1.7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Example 20) Increases SMN mRNA and Protein Production In Vitro.

(A) SMN2 splicing in SMA Type 1 fibroblasts. (B) SMN protein in SMA Type1 fibroblasts. (C) SMN protein in SMA Type 1 motor neurons. (D) SMN1 andSMN2 splicing in whole blood derived from HV. Fibroblasts from SMA Type1 patients were cultured for 24 hours (A) or 48 hours (B); motor neuronsfrom SMA Type 1 patient iPSCs (induced Pluripotent Stem Cells) werecultured for 72 hours (C) and whole blood cells from healthy volunteers(HV) for 4 hours (D) in the presence of the compound of Example 20. SMNsplicing was assessed by RT-PCR, and SMN protein levels were assessed byhomogenous time-resolved fluorescence (HTRF) in fibroblast lysates, andby immunostaining for SMN in motor neurons. Data representmeans±standard error (SEM) of 3 evaluations per data point and areexpressed as fold change vs. untreated controls.

FIG. 2.7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Example 20) Induces SMN Protein Expression In Vivo.

(A) SMN protein in brains of C/C-allele mice. (B) SMN protein in brainsof SMNΔ7 mice. (C) SMN protein in quadriceps muscle of C/C-allele mice.(D) SMN protein in quadriceps muscle of SMNΔ7 mice. C/C-allele mice andSMNΔ7 mice were treated with the compound of Example 20. One hour afterthe last dose, brains and quadriceps muscles were collected and levelsof SMN protein were assessed by HTRF. Data represent means±SEM of 5-6animals per group and are expressed as fold change vs. vehicle-treatedcontrols. *=p<0.05, **=p<0.01, ***=p<0.001 vs. untreated controls.

FIG. 3. In Vivo Effects of Treatment SMNΔ7 Mice with7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Example 20).

SMNΔ7 mice were treated with the compound of Example 20 from P3 onwardsand animal survival (A) and body weight (B) were assessed daily untilP100. Data represent means±SEM of 10-12 mice per group. HET=heterozygouslittermates.

FIG. 4. Protection of Motor Circuits and Muscle Atrophy by7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Example 20) in SMNΔ7 Mice In Vivo.

SMNΔ7 mice were treated with the compound of Example 20 from P3 to P14,and neuromuscular connectivity and muscle atrophy were assessed byimmunohistochemistry. (A) vGlut1-positive proprioceptive inputs in L3-5spinal cord. (B) Ventral motor axons in L3-5 spinal cord. (C) NMJinnervation onto longissimus muscle. (D) EDL muscle cross-sectionalarea. Data represent means±SEM of 4-5 mice per group. *=p<0.05;**=p<0.01; ***=p<0.001 vs. vehicle-treated SMNΔ7 mice.

FIG. 5. Alternative Splicing of FoxM1 In Vitro

SMA Type I patient fibroblasts were treated with the compound of Example20 for 24 hours, and FoxM1 full-length (FL) and exon 9-lacking (Δ9)mRNAs were analyzed by RT-qPCR. Data represent means±SEM of 6repetitions and are expressed as fold change vs. untreated controls.

FIG. 6. Oil-in-Water Emulsions

Photographs of composition 4A prior to (A) and immediately after theaddition of 20% (B) or 30% (C) of tartrate buffer solution (composition5A) and thereby resulting water-in-oil emulsions.

FIG. 7. Stability of Oil-in-Water Emulsions

Photographs of water-in-oil emulsions comprising 70% composition 4A and30% composition 5A 15 minutes after constitution (A) (10 times shaking)and 30 min after constitution (B) (10 times shaking).

FIG. 8. Oil-in-Water Emulsions

Photographs of composition 4F prior to (A) and immediately after theaddition of 20% (B left), 25% (B middle) or 30% (B right) of tartratebuffer solution (composition 5A) and thereby resulting water-in-oilemulsions.

FIG. 9. Stability of Oil-in-Water Emulsions

Photographs of water-in-oil emulsions comprising 70% composition 4F and30% composition 5A 15 minutes after constitution (A) (10 times shaking)and 30 min after constitution (B) (10 times shaking).

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should however not be construed as limiting thescope of the invention.

Abbreviations Used

ACN: Acetonitrile; CH₂Cl₂: dichloromethane (DCM); DIPEA: diisopropylethylamine; DMA: dimethyl acetamide; TEA: triethylamine; RT: roomtemperature; B₂(pin)₂: bis(pinacolato)diboron; Pd(dppf)Cl₂:(1,1′-Bis(diphenylphosphino)ferrocene)palladium(II) dichloride; PPTS:Pyridinium p-toluenesulfonate.

Intermediate 17-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onea) 2-chloro-7-fluoro-pyrido[1,2-a]pyrimidin-4-one

A mixture of 2-amino-5-fluoropyridine (11.20 g, 0.10 mol) and dimethylmalonate (57.0 mL, 0.50 mol) was heated at 230° C. for 1.5 h. Aftercooling to room temperature, the precipitate was filtered and washedwith ACN (3×) to give 7-fluoro-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-oneas a dark solid (14 g), which was used directly in the next step. MS m/z181.3 [M+H]⁺.

A dark mixture of crude7-fluoro-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one (14 g, ˜77 mmol) inPOCl₃ (50 mL) and DIPEA (13.3 mL, 77 mmol) was heated at 110° C. for 15hours. The solvent was removed and the dark residue was treated withice-water, washed with water (3×) and dried to give a brown solid. Thecrude brown solid was chromatographed (5% MeOH in CH₂Cl₂) to give2-chloro-7-fluoro-4H-pyrido[1,2-a]pyrimidin-4-one as a yellow solid(9.84 g, 50%, 2 steps), MS m/z 199.2 [M+H]⁺.

b)2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine

A mixture of 6-chloro-2-methylimidazo[1,2-b]pyridazine (900 mg, 5.37mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.36g, 5.37 mmol, 1.0 eq.), KOAc (1.05 g, 10.7 mmol) and Pd(dppf)Cl₂—CH₂Cl₂(393 mg, 0.54 mmol) in dioxane (50 mL) was degassed and heated under N₂at 95° C. After 15 hours, the mixture was diluted with EtOAc, filteredthrough celite and concentrated under vacuum to give2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazinewhich was used directly in the next step.

c)7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

To a solution of 2-chloro-7-fluoro-4H-pyrido[1,2-a]pyrimidin-4-one (750mg, 3.78 mmol) in ACN (36 mL) was added2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine(1.17 g, 4.53 mmol, Eq: 1.2), Pd(Ph₃P)₄ (218 mg, 0.189 mmol, 0.05 eq.)and an aqueous solution of K₂CO₃ (3.78 mL, 7.55 mmol, 2.0 eq.). Themixture was degassed and heated under argon at 105° C. overnight. Thereaction was cooled to RT, and filtered. The precipitate was washed withEt₂O and then water, dried in vacuo to give 250 mg (22%) of7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneas a light brown solid. MS m/z 296.1 [M+H]⁺.

Intermediate 22-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-onea)2,8-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine

In a sealed flask, 3,6-dichloro-4-methylpyridazine (27 g, 161 mmol) wassuspended in aqueous ammonia (25%, 300 mL). The reaction mixture washeated at 110° C. for 48 hours (turned into solution after 1 hour).After cooling to room temperature, the reaction was poured into CH₂Cl₂,and the organic phase was separated, dried over Na₂SO₄, and concentratedunder vacuum, to give 22.4 g of 6-chloro-4-methyl-pyridazin-3-amine and6-chloro-5-methyl-pyridazin-3-amine as a mixture of regioisomers whichwere used directly in the next step.

The mixture of regioisomers 6-chloro-4-methyl-pyridazin-3-amine and6-chloro-5-methyl-pyridazin-3-amine (22.4 g) was suspended in 2-propanol(300 mL). 1-bromo-2,2-dimethoxypropane (36.0 g, 26.6 mL, 193 mmol, 1.2eq.) and PPTS (2.96 g, 11.6 mmol, 0.0725 eq.) were added, and theresulting solution was heated at 105° C. overnight. The solvent wasremoved in vacuo and the residue was taken up in CH₂Cl₂ and washed withNaHCO₃. The organic phases were dried over Na₂SO₄, concentrated in vacuoand the crude light brown solid was chromatographed (EtOAc/Heptane1/2-1/1) to give separately 6.1 g of6-chloro-2,8-dimethyl-imidazo[1,2-b]pyridazine MS m/z 182.1 [M+H]⁺ (21%)as a white solid and 5.9 g of6-chloro-2,7-dimethyl-imidazo[1,2-b]pyridazine MS m/z 182.1 [M+H]⁺ (20%)as a white solid.

A mixture of 6-chloro-2,8-dimethylimidazo[1,2-b]pyridazine (0.9 g, 4.96mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.26g, 4.96 mmol, 1.0 eq.), KOAc (0.97 g, 9.91 mmol) and Pd(dppf)Cl₂.CH₂Cl₂(363 mg, 0.49 mmol) in dioxane (50 mL) was degassed and heated under N₂at 110° C. After 15 hours, the mixture was diluted with EtOAc, filteredthrough celite and concentrated under vacuum to give2,8-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazinewhich was used directly in the next step.

b)2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one

To a solution of 2-chloro-7-fluoro-4H-pyrido[1,2-a]pyrimidin-4-one (750mg, 3.78 mmol, described herein above) in ACN (36 mL) was added2,8-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine(1.24 g, 4.53 mmol, 1.2 eq.), Pd(Ph₃P)₄ (218 mg, 0.189 mmol, 0.05 eq.)and an aqueous solution of K₂CO₃ (3.78 mL, 7.55 mmol, 2.0 eq.). Themixture was degassed and heated under argon at 100° C. for 6 hours. Thereaction was cooled to RT, and filtered. The precipitate was washed withEt₂O and then water, dried in vacuo to give 700 mg (60%) of2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-oneas a light brown solid. MS m/z 310.1 [M+1-1]⁺.

Intermediate 37-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onea) 2-chloro-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one

A mixture of 5-fluoro-3-methylpyridin-2-amine (3.3 g, 26.2 mmol) anddimethyl malonate (15.0 mL, 0.13 mol, 5.0 eq.) was heated at 210° C. for1.5 hours. After cooling to room temperature, the precipitate wasfiltered and washed with ACN (3×) to give7-fluoro-2-hydroxy-9-methyl-pyrido[1,2-a]pyrimidin-4-one as a dark solid(2.3 g), which was used directly in the next step. MS m/z 195.1 [M+H]⁺.

A mixture of crude7-fluoro-2-hydroxy-9-methyl-pyrido[1,2-a]pyrimidin-4-one (2.3 g, 11.8mmol) in POCl₃ (7.7 mL, 82.9 mmol) and DIEA (2.07 mL, 11.8 mmol) washeated at 110° C. for 15 hours. The solvent was removed and the residuewas treated with ice-water, washed with water (3×) and dried to give abrown solid. The crude brown solid was chromatographed (5% MeOH inCH₂Cl₂) to give 2-chloro-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-oneas a yellow solid (1.77 g, 70% over 2 steps), MS m/z 213.1 [M+H]⁺.

b)7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

To a solution of2-chloro-7-fluoro-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (2.2 g, 10.3mmol) in ACN (80 mL) was added2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine(3.22 g, 12.4 mmol, 1.2 eq., described herein above), Pd(Ph₃P)₄ (1.20 g,1.03 mmol, 0.1 eq.) and an aqueous solution of K₂CO₃ (10.3 mL, 20.7mmol, 2.0 eq.). The mixture was degassed and heated under argon at 100°C. for 6 hours. The reaction was cooled to RT, and filtered. Theprecipitate was washed with Et₂O and then water, dried in vacuo to give1.80 g (56%) of7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneas a light brown solid. MS m/z 310.1 [M+1-1]⁺.

Intermediate 42-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one

To a solution of2-chloro-7-fluoro-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (0.98 g, 4.61mmol, described herein above) in ACN (50 mL) was added2,8-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine(1.51 g, 5.53 mmol, 1.2 eq., described herein above), Pd(Ph₃P)₄ (0.32 g,0.277 mmol, 0.06 eq.) and an aqueous solution of K₂CO₃ (4.61 mL, 9.22mmol, 2.0 eq.). The mixture was degassed and heated under argon at 100°C. for 6 hours. The reaction was cooled to RT, and filtered. Theprecipitate was washed with Et₂O and water, then dried in vacuo to give0.89 g (60%) of2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-oneas a light brown solid. MS m/z 324.4 [M+H]⁺.

Example 12-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 35 mg, 0.119 mmol) and 1-methylpiperazine (47.5 mg,0.474 mmol, 4 eq.) were stirred in DMSO (1 mL) at 120° C. overnight.LC-MS showed total convertion. The solvent was removed under highvacuum. The crude product was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 9/1) to afford the title product (25 mg, 56%) as alight yellow solid. MS m/z 376.3 [M+H+].

Example 27-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 125 mg, 0.426 mmol) and(R)-octahydropyrrolo-[1,2-a]pyrazine (160 mg, 1.27 mmol, 3 eq.) werestirred in DMSO (5 mL) at 125° C. overnight. The solvent was removedunder high vacuum. The residue was taken up in CH₂Cl₂ and washed with anaqueous saturated solution of NaHCO₃. The organic layer was separatedand dried over Na₂SO₄ and concentrated in vacuo. The crude was purifiedby column chromatography (SiO₂, CH₂Cl₂/MeOH=98/2 to 95/5) to afford thetitle product (65 mg, 38%) as a light yellow solid. MS m/z 402.5 [M+H+].

Example 37-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 200 mg, 0.647 mmol) and(S)-octahydropyrrolo-[1,2-a]pyrazine (286 mg, 2.26 mmol, 3.5 eq.) werestirred in DMSO (5 mL) at 125° C. overnight. The solvent was removedunder high vacuum. The residue was taken up in CH₂Cl₂ and washed with anaqueous saturated solution of NaHCO₃. The organic layer was separatedand dried over Na₂SO₄ and concentrated in vacuo. The crude was purifiedby column chromatography (SiO₂, CH₂Cl₂/MeOH=98/2 to 95/5) to afford thetitle product (115 mg, 43%) as a light yellow solid. MS m/z 416.3[M+H⁺].

Example 47-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 200 mg, 0.647 mmol), DIPEA (0.113 mL, 0.67 mmol, 1eq.)and (R)-octahydropyrrolo-[1,2-a]pyrazine (245 mg, 1.95 mmol, 3.0 eq.)were stirred in DMSO (2.5 mL) at 125° C. overnight. The solvent wasremoved under high vacuum. The residue was taken up in CH₂Cl₂ and washedwith an aqueous saturated solution of NaHCO₃. The organic layer wasseparated and dried over Na₂SO₄ and concentrated in vacuo. The crude waspurified by column chromatography (SiO₂, CH₂Cl₂/MeOH=98/2 to 95/5) toafford the title product (132 mg, 49%) as a light yellow solid. MS m/z416.3 [M+H⁺].

Example 57-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 90 mg, 0.291 mmol), DIPEA (0.05 mL, 0.29 mmol, 1eq.)and (S)-8a-methyloctahydropyrrolo[1,2-a]pyrazine (81 mg, 0.58 mmol, 2.0eq.) were stirred in DMSO (2.5 mL) at 125° C. overnight. The solvent wasremoved under high vacuum. The residue was taken up in CH₂Cl₂ and washedwith an aqueous saturated solution of NaHCO₃. The organic layer wasseparated and dried over Na₂SO₄ and concentrated in vacuo. The crude waspurified by column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) toafford the title product (55 mg, 44%) as a light yellow solid. MS m/z430.3 [M+H⁺].

Example 67-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 90 mg, 0.291 mmol), DIPEA (0.05 mL, 0.29 mmol, 1eq.)and (R)-8a-methyloctahydropyrrolo[1,2-a]pyrazine (81 mg, 0.58 mmol, 2.0eq.) were stirred in DMSO (2.5 mL) at 125° C. overnight. The solvent wasremoved under high vacuum. The residue was taken up in CH₂Cl₂ and washedwith an aqueous saturated solution of NaHCO₃. The organic layer wasseparated and dried over Na₂SO₄ and concentrated in vacuo. The crude waspurified by column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) toafford the title product (50 mg, 40%) as a light yellow solid. MS m/z430.4 [M+H⁺].

Example 72-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 50 mg, 0.162 mmol), and cis-2,6-dimethylpiperazine (74mg, 0.647 mmol, 4.0 eq.) were stirred in DMSO (1.5 mL) at 110° C.overnight. The solvent was removed under high vacuum. The residue wastaken up in CH₂Cl₂ and washed with an aqueous saturated solution ofNaHCO₃. The organic layer was separated and dried over Na₂SO₄ andconcentrated in vacuo. The crude was purified by column chromatography(SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (32 mg,49%) as a light yellow solid. MS m/z 404.4 [M+H⁺].

Example 82-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 33 mg, 0.107 mmol), and (S)-2-methylpiperazine (43 mg,0.427 mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 120° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (18 mg, 43%) as alight yellow solid. MS m/z 390.3 [M+H⁺].

Example 92-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 85 mg, 0.275 mmol), and (R)-2-methylpiperazine (110 mg,1.10 mmol, 4.0 eq.) were stirred in DMSO (5 mL) at 120° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (35 mg, 33%) as alight yellow solid. MS m/z 390.3 [M+H⁺].

Example 107-(1,4-diazepan-1-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 33 mg, 0.107 mmol), and 1,4-diazepane (32 mg, 0.320mmol, 3.0 eq.) were stirred in DMSO (2 mL) at 120° C. overnight. Thesolvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (20 mg, 48%) as alight yellow solid. MS m/z 390.3 [M+H⁺].

Example 112-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 50 mg, 0.169 mmol), and (S)-2-methylpiperazine (68 mg,0.677 mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 110° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (40 mg, 63%) as alight yellow solid. MS m/z 376.2 [M+H⁺].

Example 122-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 50 mg, 0.169 mmol), and (R)-2-methylpiperazine (68 mg,0.677 mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 110° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (48 mg, 75%) as alight yellow solid. MS m/z 376.3 [M+H⁺].

Example 137-(1,4-diazepan-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 50 mg, 0.169 mmol), and 1,4-diazepane (68 mg, 0.677mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 110° C. overnight. Thesolvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (41 mg, 65%) as alight yellow solid. MS m/z 376.2 [M+H⁺].

Example 147-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 50 mg, 0.169 mmol), and cis-2,6-dimethylpiperazine (77mg, 0.677 mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 110° C.overnight. The solvent was removed under high vacuum. The residue wastaken up in CH₂Cl₂ and washed with an aqueous saturated solution ofNaHCO₃. The organic layer was separated and dried over Na₂SO₄ andconcentrated in vacuo. The crude was purified by column chromatography(SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (41 mg,62%) as a light yellow solid. MS m/z 390.3 [M+H⁺].

Example 157-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 50 mg, 0.169 mmol), and(S)-octahydropyrrolo[1,2-a]pyrazine (85 mg, 0.677 mmol, 4.0 eq.) werestirred in DMSO (2 mL) at 125° C. overnight. The solvent was removedunder high vacuum. The residue was taken up in CH₂Cl₂ and washed with anaqueous saturated solution of NaHCO₃. The organic layer was separatedand dried over Na₂SO₄ and concentrated in vacuo. The crude was purifiedby column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) to afford thetitle product (36 mg, 53%) as a light yellow solid. MS m/z 402.3 [M+H⁺].

Example 167-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 50 mg, 0.169 mmol) and(S)-8a-methyloctahydropyrrolo[1,2-a]pyrazine (95 mg, 0.677 mmol, 4.0eq.) were stirred in DMSO (2 mL) at 125° C. overnight. The solvent wasremoved under high vacuum. The residue was taken up in CH₂Cl₂ and washedwith an aqueous saturated solution of NaHCO₃. The organic layer wasseparated and dried over Na₂SO₄ and concentrated in vacuo. The crude waspurified by column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) toafford the title product (45 mg, 64%) as a light yellow solid. MS m/z416.3 [M+H⁺].

Example 177-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 100 mg, 0.339 mmol) and(R)-8a-methyloctahydropyrrolo[1,2-a]pyrazine (190 mg, 1.35 mmol, 4.0eq.) were stirred in DMSO (4 mL) at 125° C. overnight. The solvent wasremoved under high vacuum. The residue was taken up in CH₂Cl₂ and washedwith an aqueous saturated solution of NaHCO₃. The organic layer wasseparated and dried over Na₂SO₄ and concentrated in vacuo. The crude waspurified by column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) toafford the title product (45 mg, 64%) as a light yellow solid. MS m/z416.3 [M+H⁺].

Example 182-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a microwave reactor,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 45 mg, 0.145 mmol), (R)-1,3′-bipyrrolidinedihydrochloride (62 mg, 0.291 mmol, 2.0 eq.) and DIPEA (0.20 mL, 1.16mmol, 8 eq.) were stirred in NMP (3 mL) at 220° C. for 1 hour. Thesolvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=98/2 to 90/10) to afford the title product (25 mg, 40%) as alight yellow solid. MS m/z 430.3 [M+H⁺].

Example 197-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 50 mg, 0.169 mmol), DIPEA (0.24 mL, 1.35 mmol, 8 eq.)and 4,7-diazaspiro[2.5]octane dihydrochloride (62.7 mg, 0.339 mmol, 2.0eq.) were stirred in DMSO (2 mL) at 125° C. for 2 days. The solvent wasremoved under high vacuum. The residue was taken up in CH₂Cl₂ and washedwith an aqueous saturated solution of NaHCO₃. The organic layer wasseparated and dried over Na₂SO₄ and concentrated in vacuo. The crude waspurified by column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) toafford the title product (22 mg, 33%) as a light yellow solid. MS m/z388.3 [M+H⁺].

Example 207-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 50 mg, 0.162 mmol), DIPEA (0.22 mL, 1.29 mmol, 4 eq.)and 4,7-diazaspiro[2.5]octane dihydrochloride (32 mg, 0.320 mmol, 3.0eq.) were stirred in DMSO (2 mL) at 130° C. for 48 hours. The solventwas removed under high vacuum. The residue was taken up in CH₂Cl₂ andwashed with an aqueous saturated solution of NaHCO₃. The organic layerwas separated and dried over Na₂SO₄ and concentrated in vacuo. The crudewas purified by column chromatography (SiO₂, CH₂Cl₂/MeOH=98/2 to 95/5)to afford the title product (12 mg, 18%) as a light yellow solid. MS m/z402.3 [M+H⁺].

Example 212-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 40 mg, 0.135 mmol), DIPEA (0.19 mL, 1.08 mmol, 8 eq.)and (R)-1,3′-bipyrrolidine dihydrochloride (58 mg, 0.271 mmol, 2.0 eq.)were stirred in DMSO (4 mL) and heated at 220° C. for 40 minutes in amicrowave. The solvent was removed under high vacuum. The residue wastaken up in CH₂Cl₂ and washed with an aqueous saturated solution ofNaHCO₃. The organic layer was separated and dried over Na₂SO₄ andconcentrated in vacuo. The crude was purified by column chromatography(SiO₂, CH₂Cl₂/MeOH=98/2 to 90/10) to afford the title product (30 mg,53%) as a light yellow solid. MS m/z 416.3 [M+H⁺].

Example 222-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 40 mg, 0.129 mmol) and 2,2-dimethylpiperazine (59 mg,0.517 mmol, 4.0 eq.) were stirred in DMSO (1.6 mL) at 130° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 9/1) to afford the title product (29 mg, 55%) as alight yellow solid. MS m/z 404.3 [M+H⁺].

Example 237-(3,3-dimethylpiperazin-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 40 mg, 0.135 mmol) and 2,2-dimethylpiperazine (62 mg,0.542 mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 130° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (26 mg, 49%) as alight yellow solid. MS m/z 390.3 [M+H⁺].

Example 242-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one(Intermediate 4; 50 mg, 0.155 mmol) and (S)-2-methylpiperazine (62 mg,0.619 mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 125° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (45 mg, 72%) as alight yellow solid. MS m/z 404.3 [M+H⁺].

Example 252-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one(Intermediate 4; 50 mg, 0.155 mmol) and (R)-2-methylpiperazine (62 mg,0.619 mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 125° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (40 mg, 70%) as alight yellow solid. MS m/z 404.3 [M+H⁺].

Example 262-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one(Intermediate 4; 50 mg, 0.155 mmol) and cis-2,6-dimethylpiperazine (70mg, 0.619 mmol, 4.0 eq.) were stirred in DMSO (2 mL) at 125° C.overnight. The solvent was removed under high vacuum. The residue wastaken up in CH₂Cl₂ and washed with an aqueous saturated solution ofNaHCO₃. The organic layer was separated and dried over Na₂SO₄ andconcentrated in vacuo. The crude was purified by column chromatography(SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (26 mg,40%) as a light yellow solid. MS m/z 418.3 [M+H⁺].

Example 272-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one(Intermediate 4; 50 mg, 0.155 mmol) and 2,2-dimethylpiperazine (35 mg,0.309 mmol, 2.0 eq.) were stirred in DMSO (2 mL) at 125° C. overnight.The solvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (36 mg, 56%) as alight yellow solid. MS m/z 418.3 [M+H⁺].

Example 287-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one(Intermediate 4; 50 mg, 0.155 mmol), DIPEA (0.21 mL, 1.24 mmol, 8 eq.)and 4,7-diazaspiro[2.5]octane dihydrochloride (57 mg, 0.309 mmol, 2.0eq.) were stirred in DMSO (2 mL) at 125° C. for 2 days. The solvent wasremoved under high vacuum. The residue was taken up in CH₂Cl₂ and washedwith an aqueous saturated solution of NaHCO₃. The organic layer wasseparated and dried over Na₂SO₄ and concentrated in vacuo. The crude waspurified by column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) toafford the title product (17 mg, 26%) as a light yellow solid. MS m/z416.3 [M+H⁺].

Example 292-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 50 mg, 0.162 mmol), TEA (0.18 mL, 1.29 mmol, 8 eq.) and(2S,6S)-2,6-dimethylpiperazine dihydrochloride (90 mg, 0.485 mmol, 3.0eq.) were stirred in DMSO (2 mL) at 140° C. overnight. The solvent wasremoved under high vacuum. The residue was taken up in CH₂Cl₂ and washedwith an aqueous saturated solution of NaHCO₃. The organic layer wasseparated and dried over Na₂SO₄ and concentrated in vacuo. The crude waspurified by column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 9/1) toafford the title product (20 mg, 30%) as a light yellow solid. MS m/z404.3 [M+H⁺].

Example 302-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one(Intermediate 2; 50 mg, 0.162 mmol), DIPEA (0.22 mL, 1.29 mmol, 8 eq.)and (S)-1,3′-bipyrrolidine dihydrochloride (103 mg, 0.485 mmol, 3.0 eq.)were stirred in NMP (2 mL) at 140° C. overnight. The solvent was removedunder high vacuum. The residue was taken up in CH₂Cl₂ and washed with anaqueous saturated solution of NaHCO₃. The organic layer was separatedand dried over Na₂SO₄ and concentrated in vacuo. The crude was purifiedby column chromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 9/1) to afford thetitle product (22 mg, 32%) as a light yellow solid. MS m/z 430.3 [M+H⁺].

Example 312-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 75 mg, 0.254 mmol), TEA (0.28 mL, 2.03 mmol, 8 eq.) and(S)-1,3′-bipyrrolidine dihydrochloride (162 mg, 0.762 mmol, 3.0 eq.)were stirred in NMP (4 mL) and heated at 220° C. for 1 hour in amicrowave. The solvent was removed under high vacuum. The residue wastaken up in CH₂Cl₂ and washed with an aqueous saturated solution ofNaHCO₃. The organic layer was separated and dried over Na₂SO₄ andconcentrated in vacuo. The crude was purified by column chromatography(SiO₂, CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (12 mg,11%) as a light yellow solid. MS m/z 416.2 [M+H⁺].

Example 327-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 75 mg, 0.254 mmol), TEA (0.28 mL, 2.03 mmol, 8 eq.) and(2S,6S)-2,6-dimethylpiperazine dihydrochloride (143 mg, 0.762 mmol, 3.0eq.) were stirred in DMSO (3 mL) and heated at 140° C. overnight. Thesolvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (10 mg, 10%) as alight yellow solid. MS m/z 390.3 [M+H⁺].

Example 339-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Intermediate 3; 250 mg, 0.808 mmol), and (S)-2-methylpiperazine (405mg, 4.04 mmol, 5.0 eq.) were stirred in DMSO (6 mL) and heated at 130°C. overnight. The solvent was removed under high vacuum. The residue wastaken up in CH₂Cl₂ and washed with an aqueous saturated solution ofNaHCO₃. The organic layer was separated and dried over Na₂SO₄ andconcentrated in vacuo. The crude was purified by column chromatography(SiO₂, CH₂Cl₂/MeOH=95/5 to 85/15) to afford the title product (135 mg,43%) as a light yellow solid. MS m/z 390.3 [M+H⁺].

Example 349-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Intermediate 3; 250 mg, 0.808 mmol), and (R)-2-methylpiperazine (405mg, 4.04 mmol, 5.0 eq.) were stirred in DMSO (6 mL) and heated at 130°C. overnight. The solvent was removed under high vacuum. The residue wastaken up in CH₂Cl₂ and washed with an aqueous saturated solution ofNaHCO₃. The organic layer was separated and dried over Na₂SO₄ andconcentrated in vacuo. The crude was purified by column chromatography(SiO₂, CH₂Cl₂/MeOH=95/5 to 85/15) to afford the title product (100 mg,32%) as a light yellow solid. MS m/z 390.3 [M+H⁺].

Example 357-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Intermediate 3; 250 mg, 0.808 mmol), and (2S,6R)-2,6-dimethylpiperazine(461 mg, 4.04 mmol, 5.0 eq.) were stirred in DMSO (6 mL) and heated at130° C. overnight. The solvent was removed under high vacuum. Theresidue was taken up in CH₂Cl₂ and washed with an aqueous saturatedsolution of NaHCO₃. The organic layer was separated and dried overNa₂SO₄ and concentrated in vacuo. The crude was purified by columnchromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 85/15) to afford the titleproduct (101 mg, 31%) as a light yellow solid. MS m/z 404.3 [M+H⁺].

Example 367-(3,3-dimethylpiperazin-1-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Intermediate 3; 250 mg, 0.808 mmol), and 2,2-dimethylpiperazine (461mg, 4.04 mmol, 5.0 eq.) were stirred in DMSO (6 mL) and heated at 130°C. overnight. The solvent was removed under high vacuum. The residue wastaken up in CH₂Cl₂ and washed with an aqueous saturated solution ofNaHCO₃. The organic layer was separated and dried over Na₂SO₄ andconcentrated in vacuo. The crude was purified by column chromatography(SiO₂, CH₂Cl₂/MeOH=95/5 to 85/15) to afford the title product (120 mg,36%) as a light yellow solid. MS m/z 404.3 [M+H⁺].

Example 377-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Intermediate 3; 125 mg, 0.404 mmol), K₂CO₃ (223 mg, 1.62 mmol, 4 eq.)and 4,7-diazaspiro[2.5]octane dihydrochloride (112 mg, 0.606 mmol, 1.5eq.) were stirred in DMA (2 mL) and heated at 130° C. overnight. Thesolvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (75 mg, 46%) as alight yellow solid. MS m/z 402.2 [M+H⁺].

Example 387-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Intermediate 3; 125 mg, 0.404 mmol), K₂CO₃ (223 mg, 1.62 mmol, 4 eq.)and (2S,6S)-2,6-dimethylpiperazine dihydrochloride (113 mg, 0.606 mmol,1.5 eq.) were stirred in DMA (2 mL) and heated at 130° C. overnight. Thesolvent was removed under high vacuum. The residue was taken up inCH₂Cl₂ and washed with an aqueous saturated solution of NaHCO₃. Theorganic layer was separated and dried over Na₂SO₄ and concentrated invacuo. The crude was purified by column chromatography (SiO₂,CH₂Cl₂/MeOH=95/5 to 90/10) to afford the title product (50 mg, 31%) as alight yellow solid. MS m/z 404.3 [M+H⁺].

Example 397-[(3R)-3-ethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube,7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(Intermediate 1; 200 mg, 0.677 mmol), K₂CO₃ (374 mg, 2.71 mmol, 4 eq.)and (R)-2-ethylpiperazine dihydrochloride (238 mg, 0.606 mmol, 1.5 eq.)were stirred in DMA (3 mL) at 100° C. for 4 days. The solvent wasremoved under high vacuum. The crude was purified by columnchromatography (SiO₂, CH₂Cl₂/MeOH=95/5 to 8/2) to afford the titleproduct (168 mg, 64%) as a light yellow solid. MS m/z 390.2 [M+H⁺].

Example 40 SMN2 Minigene mRNA Splicing RT-qPCR Assay in Cultured Cells

To describe in more detail and assist in understanding the presentdescription, the following non-limiting biological examples are offeredto more fully illustrate the scope of the description and are not to beconstrued as specifically limiting the scope thereof. Such variations ofthe present description that may be now known or later developed, whichwould be within the purview of one skilled in the art to ascertain, areconsidered to fall within the scope of the present description and ashereinafter claimed. These examples illustrate the testing of certaincompounds described herein in vitro and/or in vivo and demonstrate theusefulness of the compounds for treating of SMA by enhancing theinclusion of exon 7 of SMN2 into mRNA transcribed from the SMN2 gene.Compounds of formula (I) enhance inclusion of exon 7 of SMN2 into mRNAtranscribed from the SMN2 gene and increase levels of SMN proteinproduced from the SMN2 gene, and thus can be used to treat SMA in ahuman subject in need thereof. These examples further illustrate thetesting of certain compounds described herein in vitro and/or in vivoand demonstrate the usefulness of the compounds for enhancing theinclusion of exon 7 of SMNI into mRNA transcribed from the SMN1 gene.Accordingly, compounds of formula (I) also enhance the inclusion of exon7 of SMN1 into mRNA transcribed from the SMN1 gene and increase levelsof SMN protein produced from the SMN1 gene.

The reverse transcription-quantitative PCR-based (RT-qPCR) assay is usedto quantify the level of the full length SMN2 minigene (referred toherein by the term “FL SMN2mini”) mRNA containing SMN2 exon 7 in aHEK293H cell line stably transfected with said minigene and treated witha test compound. Materials used and respective sources are listed belowin Table 1.

TABLE 1 Materials and their respective sources used in the SMN2 minigenemRNA splicing RT-qPCR assay in cultured cells. Material Source HEK293Hcells Life Technologies, Inc. (formerly Invitrogen) Catalog No.11631-017 Cells-To-Ct lysis Life Technologies, Inc. (formerly Appliedbuffer Biosystems) part No. 4399002 DMEM Life Technologies, Inc.(formerly Invitrogen) Catalog No. 11960-044 96-well flat-bottom BectonDickinson Catalog No. 353072 plates RT-PCR Enzyme Life Technologies,Inc. (formerly Applied Mix Biosystems) part No. 4388520 RT-PCR bufferLife Technologies, Inc. (formerly Applied Biosystems) part No. 4388519AgPath-ID One- Life Technologies, Inc. (formerly Applied Step RT-PCR kitBiosystems) part No. 4387391 Thermocycler Life Technologies, Inc.(formerly Applied Biosystems) 7900HT

The SMN2-A minigene construct was prepared as described in InternationalPatent Application WO2009/151546A1 page 145 paragraph [00400] to page147 paragraph [00412](incl. FIG. 1 and FIG. 3 therein).

HEK293H cells stably transfected with the SMN2-A minigene construct(10,000 cells/well) are seeded in 200 μL of cell culture medium (DMEMplus 10% FBS, with 200 μg/mL hygromycin) in 96-well flat-bottom platesand the plate is immediately swirled to ensure proper dispersal of cellsand the formation of an even monolayer of cells. Cells are allowed toattach for 6 hours. Test compounds are serially diluted 3.16-fold in100% DMSO to generate a 7-point concentration curve. A solution of testcompound (1 μL, 200× in DMSO) is added to each cell-containing well andthe plate is incubated for 24 hours in a cell culture incubator (37° C.,5% CO₂, 100% relative humidity). 2 replicates are prepared for each testcompound concentration. The cells are then lysed in the Cells-To-Ctlysis buffer and the lysate is stored at −80° C.

Full length SMN2-A minigene and GAPDH mRNA are quantified using theprimers and probes referenced in WO2014/209841A2 on page 80 in Table 1.Primer SMN Forward A (SEQ ID NO.1) hybridizes to a nucleotide sequencein exon 7 (nucleotide 22 to nucleotide 40), primer SMN Reverse A (SEQ IDNO.2) hybridizes to a nucleotide sequence in the coding sequence ofFirefly luciferase, SMN Probe A (SEQ ID NO.3) hybridizes to a nucleotidesequence in exon 7 (nucleotide 50 to nucleotide 54) and exon 8(nucleotide 1 to nucleotide 21). The combination of these threeoligonucleotides detects only SMN1 or SMN2 minigenes (RT-qPCR) and willnot detect endogenous SMN1 or SMN2 genes.

The SMN forward and reverse primers are used at final concentrations of0.4 μM. The SMN probe is used at a final concentration of 0.15 μM. TheGAPDH primers are used at final concentrations of 0.2 μM and the probeat 0.15 μM.

The SMN2-minigene GAPDH mix (15 μL total volume) is prepared bycombining 7.5 μL of 2×RT-PCR buffer, 0.4 μL of 25×RT-PCR enzyme mix,0.75 μL of 20×GAPDH primer-probe mix, 4.0075 μL of water, 2 μL of10-fold diluted cell lysate, 0.06 μL of 100 μM SMN forward primer, 0.06μL of 100 μM SMN reverse primer, and 0.225 μL of 100 μM SMN probe.

PCR is carried out at the following temperatures for the indicated time:Step 1: 48° C. (15 min); Step 2: 95° C. (10 min); Step 3: 95° C. (15sec); Step 4: 60° C. (1 min); then repeat Steps 3 and 4 for a total of40 cycles.

Each reaction mixture contains both SMN2-A minigene and GAPDHprimers/probe sets (multiplex design), allowing simultaneous measurementof the levels of two transcripts.

The increase in the abundance of the FL SMN2mini mRNA relative to thatin cells treated with vehicle control is determined from real-time PCRdata using a modified ΔΔCt method (as described in Livak and Schmittgen,Methods, 2001, 25:402-8). The amplification efficiency E is calculatedfrom the slope of the amplification curve for FL SMN2mini and GAPDHindividually. The abundance of FL SMN2mini and GAPDH mRNA are thencalculated as (1+E)^(−Ct), where Ct is the threshold value for eachamplicon. The abundance of FL SMN2mini mRNA is normalized to GAPDH mRNAabundance. The normalized FL SMN2mini mRNA abundance from testcompound-treated samples is then divided by normalized FL SMN2mini mRNAabundance from vehicle-treated cells to determine the level of FLSMN2mini mRNA relative to vehicle control.

Table 2 provides EC_(1.5×) concentrations for production of full lengthSMN2 minigene mRNA that was obtained from the 7-point concentration datagenerated according to the above procedure for particular compounds ofthe present invention.

Particular compounds of the present invention exhibit an EC_(1.5×)concentration for production of full length SMN2 minigene mRNA≤1 μM.

More particular compounds of the present invention exhibit an EC_(1.5×)concentration for production of full length SMN2 minigene mRNA ≤0.1 μM.

Most particular compounds of the present invention exhibit an EC1.5×concentration for production of full length SMN2 minigene mRNA ≤0.02 μM.

TABLE 2 EC_(1.5x) concentrations for production of full length SMN2minigene mRNA. Example EC_(1.5x) minigene (nM) 1 3.5 2 3.8 3 3.2 4 1.8 50.6 6 2.8 7 3.7 8 0.3 9 0.1 10 6.4 11 1.4 12 1.2 13 5 14 4.1 15 4 16 1.117 6.4 18 3.6 19 10.2 20 4.3 21 9.6 22 0.9 23 3.4 24 0.4 25 0.5 26 32727 39.9 28 5 29 0.3 30 3 31 6.7 32 1.6 33 0.5 34 0.9 35 4.7 36 5 37 4.438 0.3 39 0.9

Example 41 SMN Protein Assay in Cultured Cells

The SMN HTRF (homogeneous time resolved fluorescence) assay is used toquantify the level of SMN protein in SMA patient fibroblast cellstreated with test compounds. Materials used and respective sources arelisted below in Table 3.

TABLE 3 Materials and their respective sources used in the SMN proteinassay in cultured cells. Material Source SMA Type 1 human GM03813(Coriell Institute) cells Protease inhibitor Roche Applied ScienceCatalog No. cocktail 11836145001 Anti-SMN d2 Blue cap Cisbio Catalog No.63IDC002-SMN Anti-SMN kryptate Red cap Cisbio Catalog No. 63IDC002-SMNSMN reconstitution Cisbio Catalog No. 63IDC002-SMN-Buffer buffer DMEMLife Technologies (formerly Invitrogen) Catalog No. 11960-044 RIPA LysisBuffer 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% ThermoScientific NP-40 Surfact- Amps Detergent Solution (Fisher Scientific,Pittsburgh/PA), 1% Sodium deoxycholate Diluent Buffer 20 mM Tris-HCl pH7.5, 150 mM NaCl Envision Plate Perkin Elmer Model # 2103 Reader

Cells are thawed and cultured in DMEM-10% FBS for 72 hours. Cells aretrypsinized, counted and re-suspended to a concentration of 25,000cells/mL in DMEM-10% FBS. The cell suspensions are plated at 5,000 cellsper well in a 96 well microtiter plate and incubated for 3 to 5 hours.Test compounds are serially diluted 3.16-fold in 100% DMSO to generate a7-point concentration curve. 1 μL of test compound solution istransferred to cell-containing wells and cells are incubated for 48hours in a cell culture incubator (37° C., 5% CO₂, 100% relativehumidity). Triplicate samples are set up for each test compoundconcentration. After 48 hours, the supernatant is removed from the wellsand 25 μL, of the RIPA lysis buffer, containing protease inhibitors, isadded to the wells and incubated with shaking at room temperature for 1hour. 25 μL, of the diluent is added and then 35 μL, of the resultinglysate is transferred to a 384-well plate, where each well contains 5 μLof the antibody solution (1:100 dilution of anti-SMN d2 and anti-SMNkryptate in SMN reconstitution buffer). The plate is centrifuged for 1minute to bring the solution to the bottom of the wells, then incubatedovernight at room temperature. Fluorescence for each well of the plateat 665 nm and 620 nm is measured on an EnVision multilabel plate reader(Perkin-Elmer).

The normalized fluorescence signal is calculated for each sample, Blankand vehicle control well by dividing the signal at 665 nm by the signalat 620 nm. Normalizing the signal accounts for possible fluorescencequenching due to the matrix effect of the lysate. The ΔF value (ameasurement of SMN protein abundance as a percent value) for each samplewell is calculated by subtracting the normalized average fluorescencefor the Blank control wells from the normalized fluorescence for eachsample well, then dividing this difference by the normalized averagefluorescence for the Blank control wells and multiplying the resultingvalue by 100. The ΔF value for each sample well represents the SMNprotein abundance from test compound-treated samples. The ΔF value foreach sample well is divided by the ΔF value for the vehicle controlwells to calculate the fold increase in SMN protein abundance relativeto the vehicle control. Table 4 provides EC_(1.5×) concentrations forSMN protein expression that was obtained from the 7-point concentrationdata generated according to the above procedure for particular compoundsof the present invention.

Particular compounds of the present invention exhibit an EC_(1.5×)concentration for SMN protein expression ≤1 μM.

More particular compounds of the present invention exhibit an EC_(1.5×)concentration for SMN protein expression ≤100 nM.

Most particular compounds of the present invention exhibit an EC_(1.5×)concentration for SMN protein expression ≤30 nM.

Table 5 provides the maximum fold increase of SMN protein that wasobtained from the 7-point concentration data generated according to theabove procedure for particular compounds of the present invention

Particular compounds of the present invention exhibit a maximum foldincrease ≥1.5.

More particular compounds of the present invention exhibit a maximumfold increase ≥1.7.

Most particular compounds of the present invention exhibit a maximumfold increase ≥1.8.

TABLE 4 EC_(1.5x) concentrations for SMN protein expression. ExampleEC1.5x SMN protein (nM) 1 10.8 2 19.8 3 25.6 4 15.7 5 4.1 6 11 7 15.5 85.9 9 2.5 10 22.8 11 7 12 7.5 13 3 14 17.6 15 21.2 16 3 17 20.2 18 25 1929.8 20 37 21 68.7 22 13.8 23 23.9 24 4.7 25 11.9 26 1230 27 126.5 2849.7 29 2.1 30 13.6 31 27.7 32 4 33 4 34 4.4 35 19.5 36 34.4 37 45 383.1 39 15.8

TABLE 5 Maximum fold increase of SMN protein. Example max. fold increase1 1.84 2 1.76 3 1.81 4 1.76 5 1.71 6 1.84 7 1.76 8 1.85 9 1.92 10 1.9511 1.9 12 1.77 13 1.91 14 1.86 15 1.94 16 1.83 17 1.98 18 1.75 19 1.8320 1.72 21 1.54 22 1.69 23 1.63 24 1.77 25 1.79 26 1.52 27 1.57 28 1.7229 1.81 30 1.84 31 1.65 32 1.88 33 1.82 34 1.89 35 1.79 36 1.77 37 1.8738 1.85 39 1.81

Example 42 In Vitro Assay of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Example 20)

The compound of Example 20 is an orally available small molecule SMN2splicing modifier for the treatment of SMA. It has been found, that thecompound of Example 20 effectively corrects the dysfunctional splicingof the human SMN2 pre-mRNA in cultured patient cells (SMA Type 1fibroblasts) by shifting the balance of the alternative splicingreaction completely towards the inclusion of SMN2 exon 7 and theproduction of the full-length mRNA (FIG. 1A: EC₅₀ 29±8 nM for FL, 12±1nM for Δ7 mRNA). Treating cells expressing the SMN2 minigene withincreasing concentrations of compound of Example 20 resulted in adose-dependent increase in the amount of the SMN2 minigene full lengthmRNA. EC1.5× was 4.7±0.7 nM and the maximum induction was 20-fold. Theminigene assay results confirm that compound of Example 20 is a potentSMN2 splicing modifier.

To investigate SMN protein production as a consequence of alternativesplicing, an in vitro assay was performed to assess the levels of SMNprotein in fibroblasts and in spinal motor neurons derived from SMApatient iPSCs (induced Pluripotent Stem Cells) (EC50 of 12±3 nM, andEC₅₀ 182±114 nM, respectively). The maximal increase in SMN proteinabove untreated cells resulted in levels similar in both cell types(60-80%; FIGS. 1B and 1C), suggesting that in different cell types fromSMA patients, compound of Example 20 increases SMN protein levelsimilarly as a result of correcting the dysfunctional SMN2 splicing invitro.

To further assess SMN2 splicing as a potential blood biomarker, an exvivo assay was developed using whole blood cells from healthy volunteersin which SMN1 and SMN2 splicing was assessed after 4 hours treatmentwith the compound of Example 20 (at this time point, maximal splicingchanges were achieved). Whereas SMN1 splicing was largely unaffected,SMN2 splicing was dose-dependently altered towards inclusion of exon 7(FIG. 1D). Effects on splicing were evident at concentrations above 100nM of compound of Example 20, suggesting that these levels in the bloodare required to observe the in vivo pharmacodynamic (PD) effects on SMN2splicing with this assay.

Example 43 In Vivo Assay of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Example 20)

In vivo, the compound of Example 20 increases SMN protein in the brainand muscle in the severe SMNΔ7 model and the milder C/C-allele model,carrying human SMN2 transgenes. Adult C/C-allele mice were treated for10 days with vehicle or the compound of Example 20 (1, 3 or 10 mg/kg PO,daily), and 3 days old (P3) SMNΔ7 mice were treated for 7 days withvehicle or the compound of Example 20 (0.1, 0.3, 1 or 3 mg/kg IP,daily). The compound of Example 20 dose-dependently increased SMNprotein levels in brain and muscle tissue, with a maximum effect of a2-3 fold increase reached at 10 mg/kg in adult C/C-allele mice and at1-3 mg/kg in neonatal SMNΔ7 mice (FIG. 2). Thus, in the muscle ofC/C-allele mice at the 10 mg/kg dose, the SMN levels achieved were nodifferent from those in heterozygous mice. In SMNΔ7 mice, the SMNprotein increase was only partial in both brain and muscle, reachingapproximately 43% (brain) and 55% (muscle) of protein levels inheterozygous mice. These data demonstrate that the compound of example20 increases SMN protein in both brain and muscle tissues of transgenicmouse models of SMA.

Functional benefits were assessed in the severe and mild SMA mousemodels. SMNΔ7 mice were treated from P3 to P23 once daily by IPinjection of vehicle or the compound of Example 20, and from P24 onwardsonce daily by oral gavage. During the treatment period, body weight andanimal survival were monitored. Over the 100-day observation period,only two heterozygous littermates died. In contrast, all vehicle-treatedmice died before P21 with a median survival time (MST) of 10.5 days.Example 20 treatment dose-dependently prolonged animal survival (FIG.3A). A minor but significant prolongation of MST to P26 was observed ata lower dose (0.1 mg/kg IP until P23 and 0.3 mg/kg PO thereafter). Themid-(0.3 mg/kg IP through P23 and 1 mg/kg PO thereafter), mid/high-(1mg/kg IP through P23 and 3 mg/kg PO thereafter), and high-dose (3 mg/kgIP through P23 and 10 mg/kg PO thereafter) treatment groups resulted in80%, 82%, and 73% respectively, of animals surviving up to P100, nodifferent from heterozygous littermates with 83% surviving at P100.

Body weight increase of SMNΔ7 mice throughout the study was severelyimpaired and only mildly corrected by the low dose of the compound ofExample 20. Treatment with the mid-, mid/high-, and high-doses of thecompound of Example 20 resulted in a 71%, 82%, and 85% respectively,recovery in body weight gain as compared to heterozygous littermatesthat do not show any SMA-related phenotype (FIG. 3B). These data suggestthat treatment with the compound of Example 20 dose-dependently preventsthe manifestation of the SMA phenotype in the severely affected SMNΔ7mice when dosing is started at P3.

Lastly, the compound of Example 20 improves neuromuscular connectivityin a severe SMA mouse model in vivo. SMNΔ7 mice were treated from P3 toP14 by IP application of vehicle or 0.1, 0.3, 1 mg/kg Example 20 onceper day. At P14, 1 hour after the last dose spinal cord and muscletissues were processed for histological assessment. Relative toheterozygous littermates, SMNΔ7 mice showed a significant loss ofvesicular glutamate transporter 1 (vGlut1) proprioceptive motor neuroninputs, loss of motor axons, neuromuscular junction (NMJ) denervation inlongissimus muscle, and muscle atrophy. Example 20 treatmentdose-dependently and significantly increased the number of vGlut1inputs, the number of motor axons, the percentage of fully innervatedNMJs and the fiber size in extensor digitorum longus (EDL) musclesrelative to vehicle-treated SMNΔ7 mice (FIG. 4). These data suggest thattreatment with the compound of Example 20, when started at P3, protectsboth central and peripheral aspects of NMJ denervation and protectsagainst muscle atrophy in severely affected SMNΔ7 mice.

Example 44 Transcriptional Profiling Analysis of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(Example 20)

To identify other potential genes alternatively spliced by the compoundof Example 20, a transcriptional profiling analysis was performed whichrevealed that the splicing events of a few genes were also affected attherapeutically relevant concentration of 121 nM (EC₉₀, 10-fold higherthan EC₅₀): STRN3, SLC25A17 and GGCT compared to control. The specificfunction of STRN3, SLC25A17, and GGCT and consequences of theirdysregulation have not been elucidated so far. These three genes arealso consistently found to be affected at the higher dose (5-fold higherthan the EC₉₀), a dose used in order to illustrate the maximum effect ofthis compound. Splicing events of 11 genes, including the genes FoxM1and MADD, were affected at the higher dose but not by the lower dose.FoxM1 and MADD have been described as being involved in cell cycleregulation and apoptosis, respectively. A recent report on SMN2 splicingmodifiers, suggests that the compound of Example 20 is relativelyspecific compared with another molecule, NVS-SM1, for which there were39 candidate events where splicing changed in response to treatment[Palacino et al, Nat Chem Biol. 2015 July; 11(7):511-7].

In addition, the transcriptional profiling analysis demonstrated thatthe expression levels of 0 genes were changed (p<0.01) upon treatmentwith the compound of Example 20 at the 121 nM dose. These datademonstrate the relative specificity of the compound compared withpublished data on the expression level changes of another alternativeSMN2 splicing molecule, NVS-SM1, in which there were 175 genes changedat greater than ±2-fold (p<0.05), and NVS-SM3 which significantlyaltered 23 genes [Palacino et al, Nat Chem Biol. 2015 July;11(7):511-7].

FoxM1, a gene alternatively spliced by other splicing modifiercompounds, encodes a cell cycle regulator. In humans and higher primatesonly, the transcriptionally inactive FoxM1a variant contains exon 9 (FL)and the transcriptionally active FoxM1b/c variants lack exon 9 (A9) [Yeet al, Future Oncol. 2007 February; 3(1):1-3. Laoukili et al, BiochimBiophys Acta. 2007 January; 1775(1):92-102]. Using RT qPCR with specificprimers for FoxM1a (FL), and FoxM1b/c (Δ9), the modification ofalternative splicing of FoxM1 after Example 20 treatment was confirmed(EC₅₀ 67±32 nM for FL, 139±43 nM for Δ9 mRNA; see FIG. 5). Increasedexpression of the FoxM1A isoform, together with decreased expression ofFoxM1 isoforms lacking exon 9, has the capability to disturb and inhibitcell cycle progression if splicing changes are at a level that isbiologically significant. Thus, the compound of Example 20 acts in asimilar way on the SMN2 and FoxM1 splicing machinery, but with opposingoutcomes with regard to protein function and at varying degrees. TheEC₅₀ for MADD, a gene also identified as affected by splicing modifiersincluding the compound of Example 20 at high concentrations and known tobe involved in apoptotic processes, is not known.

Example 46 Pharmaceutical Compositions Comprising Olesoxime

Examples of compositions comprising olexosime are described inUS2010099652A1. Olesoxime is stable in the solid state (>36 months at25° C./60% RH), exhibiting no change in purity profile under long-termand accelerated stress conditions. Olesoxime has low aqueous solubility(less than 5 μg/ml) across the physiological pH range. It is freelysoluble or soluble in a range of non-aqueous solvents.

In order to provide an age-appropriate formulation for a wide age range,an oral liquid composition has been developed. It has been found that aparticularly beneficial pharmaceutical composition can be achieved bypreparing an oral or gastric suspension of olexosime powder in sesameoil due to superior stability performance. Olesoxime solubility insesame oil (approximately 35 mg/ml) is insufficient to enable thepreparation of a solution while limiting the amounts of oil absorbed bythe subject. Palatability (taste, smell and texture) is acceptablewithout further excipients.

7.5 g crystalline olesoxime powder (particle size distribution with d90value 70-90 μm) was suspended in 75 ml (61.6 g) sesame oil (refined) asvehicle through agitation (e.g. shaking) to yield a homogeneoussuspension (final olesoxime concentration 100 mg/ml). Such oralsuspension has been found to be stable for at least 3 months at 25°C./60% RH regarding degradation, appearance, color, content uniformityand microbial limits.

Example 45 Oral Solutions Comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one,the compound of Example 20, can be formulated as oral aqueous solutionby dissolving the drug substance in a buffer system at pH of less thanpH4, particularly pH 3.4, in order to provide sufficiently high drugconcentration, e.g. citric buffer, malate, maleate, or tartrate buffer,more particularly malate or tartrate, most particularly tartrate buffer.

Long term stability of formulations of the compound of Example 20 bypreparing a dry powder or granulation for constitution of an oralsolution. Buffer system can be incorporated into dry formulation by theselection of organic acid and salts thereof as fine crystalline powders,e.g. tribasic sodium citrate dihydrate and citric acid anhydrous, sodiummalate and malic acid, or preferably potassium sodium tartrate andtartaric acid.

Powders or granules comprising the compound of Example 20 may comprise aextragranular filler, such as sorbitol, isomalt, or mannitol, andcombinations thereof, which ensure fast dissolution of the powder blendduring constitution of the oral solution. In introduction of a diluentthe powder blend can be granulated by dry compaction in order to improvethe flowability and to ensure robust uniformity.

Ingredients for the constitution of a solvent system for the compound ofExample 20 can be formulated as separate formulation. The constitutedsolvent can be used for dissolution of the compound of Example 20 in abottle at the start of the in-use period of the oral solution.

The constituted oral solution of the compound of Example 20 in a buffercan be can provide in-use times of more than 2 weeks by the use ofstabilizers and antioxidants, such as vitamin E TPGS, disodium edetate,butyl hydroxyl toluol, riboflavin, or preferably ascorbic acid, and incombinations thereof.

Table 6 provides a number of oral solutions providing stability insolution of more than 2 weeks.

TABLE 6 Oral solutions of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one at concentration of 0.1 mg/ml, 1.0mg/ml and 3.0 mg/ml. Composition 1A Composition 1B Composition 1CComposition 1D 0.1 mg/ml 1.0 mg/ml 3.0 mg/ml 1.0 mg/ml Ingredients (mg)(mg) (mg) (mg) 7-(4,7-diazaspiro[2.5]octan- 20.0 200.0 600.0 2007-yl)-2-(2,8-dimethylimidazo[1,2- b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one citric acid anhydrous 1077.2 1077.2 1921.2 — sodiumcitrate dihydrate 115.6 115.6 0.0 — Tartaric Acid anhydrous — — — 1274.0Potassium Sodium Tartrate x4H₂O — — — 347.6 ascorbic acid 70.5 70.5211.5 70.5 disodium edetate 33.6 33.6 100.8 33.6 water for injection ad200.0 ml ad 200.0 ml ad 200.0 ml ad 200.0 ml

Example 46 Powder Blends as Vehicles for Constitution of Oral Solutionsof7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

Table 7 represents a granulated powder blend for the constitution of asolvent, which is suitable to dissolve7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one,and to obtain an oral solution at pH3.5 which is stable for more than 2weeks. The blend contains polyethylene glycol 6000 as water solublelubricant, sodium benzoate as preservative, sucralose as sweetener, andstrawberry flavor for the purpose of improving the taste, particularlyfor use in pediatric patients.

The compositions of Table 7 together with 80 ml water provideconstitution solvents suitable for the dissolution of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(80 mg, 240 mg and 400 mg respectively).

TABLE 7 Powder blend of a vehicle for constitution of an oral solutionof 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one at pH 3.4 with API concentrations of 1.0, 3.0 and 5.0 mg/ml.Dedicated to concentration of 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2- b]pyridazin-6-yl)pyrido[1,2-Composition 2A Composition 2B Composition 2C a]pyrimidin-4-one insolution: 1 mg/ml (mg) 3 mg/ml (mg) 5 mg/ml (mg) intragranular: Mannitol1′525.78 1′554.58 1′566.58 Tartaric Acid 148.00 180.00 194.00 PotassiumSodium Tartrate *4H₂O 173.60 112.80 86.80 Sodium Benzoate micronized80.00 80.00 80.00 Ascorbic Acid fine powder 28.18 28.18 28.18 DisodiumEdetate 13.44 13.44 13.44 PEG 6000 25.00 25.00 25.00 Sucralose 16.0016.00 16.00 Total intragranular: 2′010.0 2′010.0 2′010.0 extragranular:Mannitol 160C 250.00 250.00 250.00 Strawberry flavor 240.00 240.00240.00 Total: 2′500.0 2′500.0 2′500.0

Example 47 Powder Blends Comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onefor Constitution of Oral Solutions

Table 8 represents an oral solutions comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onewhich have been constituted by the use of constituted vehicle solutionfrom example 46 for the dissolution of the active compound. The vehicleis suitable for constitution of an oral solution at pH3.4 which isstable for more than 2 weeks. The compositions of Table 8 together with80 ml water provide oral solutions comprising 1 mg/ml, 3 mg/ml resp. 5mg/ml of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one.

TABLE 8 Oral solution constitution of an oral solution comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one at pH 3.5 with API concentrations of 1.0, 3.0 and 5.0 mg/ml.Composition 2A Composition 2B Composition 2C 1 mg/ml 3 mg/ml 5 mg/ml(mg) (mg) (mg) 7-(4,7-diazaspiro[2.5]octan- 80 240 4007-yl)-2-(2,8-dimethylimidazo[1,2- b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one Mannitol 1′525.78 1′554.58 1′566.58 Tartaric Acid148.00 180.00 194.00 Potassium Sodium Tartrate *4H₂O 173.60 112.80 86.80Sodium Benzoate micronized 80.00 80.00 80.00 Ascorbic Acid fine powder28.18 28.18 28.18 Disodium Edetate 13.44 13.44 13.44 PEG 6000 25.0025.00 25.00 Sucralose 16.00 16.00 16.00 Mannitol 160C 250.00 250.00250.00 Strawberry flavor 240.00 240.00 240.00 Water ad 80 ml ad 80 ml ad80 ml Total: 80 ml 80 ml 80 ml

Example 48 Powder Blends of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onefor constitution of oral solutions

Table 9 provides powder blends comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onewhich may be used to constitute oral solutions together with 90 mlwater. The compositions of Table 9 may also be constituted from solventprepared from a vehicle powder blend (similar to example 46) followed bydissolution of API.

TABLE 9 Oral solutions of 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin- 4-one atconcentration of 1.0 mg/ml in a bottle containing 90 ml. Composition 3AComposition 3B (mg) (mg) 7-(4,7-diazaspiro[2.5]octan- 90.0 90.07-yl)-2-(2,8-dimethylimidazo[1,2- b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one Mannitol 1200.0 1200.0 Maltodextrin — 450.0 Lactose450.0 — D-L tartaric acid 573.3 573.3 Disodium tartrate dihydrate 156.4156.4 Ascorbic acid 31.7 31.7 Disodium edetate * 4H₂O 15.1 15.1Sucralose 18.0 — Sodium saccharin 18.0 Sodium benzoate 90.0 — Sorbicacid — 90.0 PEG 6000 18.0 — Strawberry flavor 180.0 Vanilla flavor —180.0 Total per bottle (mg): 2822.5 2804.5

Example 49 Powder Blends of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onefor Constitution of Oral Solutions

Table 10 provides powder blends comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onewhich may be used to constitute oral solutions together with 80 mlwater. The compositions of Table 10 may also be constituted from solventprepared from a vehicle powder blend (similar to example 46) followed bydissolution of API.

TABLE 10 Powder blend for the preparation of an oral solution of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one at concentration of 1mg/ml in a bottle containing 80 ml water. Quantity percentage per bottlesolids (mg) (%) intragranular: 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-80.00 3.20 (2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one Mannitol (Parteck M100) 1445.94 57.84D-L tartaric acid 147.68 5.91 Potassium sodium tartrate 173.76 6.95Sodium benzoate 80.00 3.20 Ascorbic acid 28.18 1.13 Disodium edetate13.44 0.54 Sucralose 16.00 0.64 PEG 6000 25.00 1.00 Total Dry: 2010.0080.40 extragranular: Strawberry flavor PHS-180152 240.00 10.00 Mannitol160C 250.00 9.60 Total per bottle (mg): 2500.00 100.00

Example 50 Stability of Oral Solutions of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

Table 11 provides a comparison of the stabilities of various solutionsof7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one,expressed as API purity in percent. The API has been found to be stablein all oral solutions investigated without remarkable degradation overseveral weeks at ambient temperature as well at 5° C.

Composition 1A of Example 45 comprises 0.1 mg/ml of API in a citratebuffer system together with ascorbic acid as antioxidant and disodiumedetate as stabilizer.

Composition 2A of Example 46 was constituted with 200 ml of water todissolve 200 mg of API (1 mg/ml).

Compositions 3A and 3B of Example 48 were constituted from API powderblend together with 90 ml of water (1 mg/ml).

TABLE 11 Stability of solution of various compositions stored at 5° C.or 25° C. in amber glass bottles. Purity of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (%) after 7 days after 17 days after 17days Composition initial at 25° C. at 5° C. at 25° C. 1A 99.29 99.1799.26 — 2A 99.33 99.23 99.29 — 3A 99.32 99.20 99.29 — 3A 99.34 — — 99.283B 99.33 — — 99.21

Example 51 Water-in-Oil Emulsions Comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneand Olesoxime

For a combined dosing of the compound of Example 20 together witholesoxime with one single composition the aqueous oral solutions of thecompound of Example 20 can be combined with an oily solution ofolesoxime by the constitution of an oral suspension. An oily solution ofolesoxime (as e.g. in Example 46) can be transferred into a bottlecontaining the constituted solution of the compound of Example 20 andsubsequently an emulsion can be formed by manually shaking the closedbottles for 5-20 times, preferably 10 times. The oily solution ofolesoxime is a solution in sesame oil which may contain emulsifyingand/or lipophilic solubilizing agents such as glyceryl mono-oleate(Peceol™, Inwitor 948™, Capmul GMO™), glyceryl mono-linoleate (Maisine35-1™), sorbitan mono-oleate (Span 80™), or oleic acid, to increase thesolubility of olesoxime in the oily solvent and to enable the formationof an emulsion from the oily solution when it will be combined with thesolutions of the compound of Example 20. Emulsifiers and solubilizingagents which are dispersed in the oily solvent, optionally prior todissolution of olesoxime with the application of heat, can be combinedwith more polar surfactants with a HLB value of less than 7, e.g.polysorbate 80 (Tween 80™), caprylocaproyl polyoxyl glycerides(Labrasol™) in order to provide an emulsion of higher dispersibility andlonger physical stability after constitution. The emulsion can haveeither the aqueous phase or the oily phase as dispersed inner phase independence of the selected ratio between the lipophilic surfactant withlow HLB and the more hydrophilic surfactant with high HLB value.

Table 12 provides 2 examples for an oily vehicle which is suitable forthe formation of an water-in-oil emulsion, comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-onein the aqueous phase and olesoxime in the lipidic phase.

TABLE 12 Oily vehicles for Olesoxime to constitute a water-in-oilemulsion with7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one oral solution. Composition4A Composition 4F Ingredients (%) (%) Sesame oil 90.0 90.0 Sorbitanmonooleate 7.0 Peceol ™ (Glyceryl monooleate) 6.1 Polysorbate 80 3.0Labrasol ™ (caprylocaproyl 3.9 polyoxyl glicerides)

With composition 4A and 4F up to 30% (w/w) aqueous tartrate buffersolution pH3.3 could be dispersed as emulsion after 10 times shaking.The emulsions were visually homogeneous for at least 15 minutes.

Table 3 (Composition 5A) represents a solution used for preparing anaqueous solution of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one.

TABLE 13 Aqueous solvent for 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one.Composition 5A Ingredients: mg Tartaric Acid 184.6 Potassium SodiumTartrate x4H2O 217.2 Ascorbic Acid 35.23 Disodium Edetate anhydrous16.81 Purified water Ad 100 ml

FIG. 6 provides photographs of composition 4A prior to (A) andimmediately after the addition of 20% (B) or 30% (C) of tartrate buffersolution (composition 5A) and thereby resulting water-in-oil emulsions.

FIG. 7 provides photographs of water-in-oil emulsions comprising 70%composition 4A and 30% composition 5A 15 minutes after constitution (A)(10 times shaking) and 30 min after constitution (B) (10 times shaking).

FIG. 8 provides photographs of composition 4F prior to (A) andimmediately after the addition of 20% (B left), 25% (B middle) or 30% (Bright) of tartrate buffer solution (composition 5A) and therebyresulting water-in-oil emulsions.

FIG. 9 provides photographs of water-in-oil emulsions comprising 70%composition 4F and 30% composition 5A 15 minutes after constitution (A)(10 times shaking) and 30 min after constitution (B) (10 times shaking).

All emulsions prepared were stable for at least 30 minutes.

Example 52 Oily Solutions comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyridopyrimidin-4-one and Olesoxime

Alternatively to emulsions, co-formulations of the compound of Example20 and olesoxime can be prepared by dissolving both drug substances inan oily solvent containing sesame oil and lipophilic surfactants, suchas glyceryl mono-oleate (Peceol™, Inwitor 948™, Capmul GMO™), glycerylmono-linoleate (Maisine 35-1™), sorbitan mono-oleate (Span 80™), oroleic acid, to enable improved solubility in the solvent.

Table 14 provides an oily solvent system which provides increasedsolubility for both drugs and leads to sufficient stability for anin-use time after constitution as shown in Table 15.

TABLE 14 Oily solution of 100 mg/ml Olesoxime and 10 mg/ml 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one. Ingredients Composition6A Composition 6B 7-(4,7-diazaspiro[2.5]octan- 10.0 mg 10.0 mg7-yl)-2-(2,8-dimethylimidazo[1,2- b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one Olesoxime 100.0 mg 100.0 mg Butyl hydroxanisol 18.9 mg— (0.02% w/w) Vitamin E — 236.25 mg (0.25% w/w) Oleic acid 9.45 g 9.45 g(10% w/w) (10% w/w) Maisine ™ ad 100.0 ml ad 100.0 ml GlycerylMonolinoleate

TABLE 15 Stability of oily solution compositions 6A and 6B. after 1 dayafter 7 days at room after 7 days at room Composition initialtemperature at 4° C. temperature Content7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (%) 6A 99.5 97.0 99.0 96.56B 99.5 95.4 98.7 96.3 Content Olesoxime (%) 6A 98 98 98 98 6B 98 98 9897

Example 53 Powder Blends of Vehicles and Stability of Oral SolutionsConstituted Therefrom

Table 16 provides dry granulated powder blends of vehicles suitable forconstitution of oral solutions comprising7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(e.g. 0.25 or 1.5 mg/ml) at pH 3.4.

In these compositions the exact amount of tartaric acid required toreach the target pH has been used instead of buffer consisting of theacid and the corresponding salt. In-use stability of the solution for atleast 17 days could be demonstrated as can be seen from Table 17.

TABLE 16 Dry granulated powder blends of vehicles for constitution.Composition 7a Composition 7b (Vehicle for (Vehicle for 0.25 mg/ml; 1.5mg/ml; Ingredients mg per bottle Mannitol 2019.93 1948.63 Tartaric acid92.00 163.30 Sodium benzoate 64.00 64.00 Ascorbic acid 28.18 28.18Polyethylene glycol 6000 25.00 25.00 disodium edetate 14.89 14.89sucralose 16.00 16.00 Strawberry flavor 240.00 240.00 total per bottle(mg) 2500.0 2500.0

TABLE 17 Stabilities of oral solutions comprising circa 0.25 mg/ml orcirca 1.5 mg/ml of 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin- 4-one(indicated as API in the table) in vehicle solution of Composition 7a or7b constituted with 80 ml water for injection. 5° C. 25° C. Vehicle APIcontent API Purity API content API Purity days Composition [mg/ml] [%][mg/ml] [%] 0 7a 0.24 99.56 0.24 99.56 10 0.24 99.57 0.24 99.55 17 0.2499.60 0.24 99.50 0 7b 1.42 99.56 1.43 99.54 10 1.45 99.57 1.43 99.56 171.45 99.50 1.45 99.50

Example 54 Powder Blends of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneand Stability of Oral Solutions Constituted Therefrom

The dry granulated powder blends of Composition 8a, 8b, 8c and 8d ofTable 18 already include the API in order to simplify the constitutionprocedure for the solution. Compositions 8a to 8d exhibit reduced powderfill weight and contain isomalt as second diluent in order to improvethe granule properties. Excellent stability up to one month in solutioncould be demonstrated with both—water for injection and potable water—ascan be seen from Table 19.

TABLE 18 Dry granulated powder blends for constitution of oral solutionsof 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one(0.25 mg/ml or 0.75 mg/ml) constituted with 80 ml water for injection atpH 3.4. Composition 8a Composition 8b Composition 8c Composition 8dIngredients mg per bottle 7-(4,7-diazaspiro[2.5]octan- 20.0 60.0 20 207-yl)-2-(2,8-dimethylimidazo[1,2- b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one Mannitol 514.2 474.25 364.19 1373.99 Isomalt 90.7 83.764.27 242.47 Tartaric Acid fine powder 92.0 120.5 92.00 92.00 SodiumBenzoate 64.0 64.0 64.00 64.00 micronized Ascorbic Acid fine powder 28.214.1 14.09 14.09 Sucralose 16.0 16.0 16.00 16.00 Disodium edetate *2H₂O14.9 7.45 7.45 7.45 PEG 6000 10.0 10.0 8.00 8.00 Strawberry flavor 150.0150.0 150 150 total 1000.0 1000.0 800.0 2000.0

TABLE 19 Stabilities of oral solutions comprising circa 0.25 mg/ml of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (indicated as API in the table) invehicle solution of Composition 8a constituted with 80 ml water forinjection (i.e. double distilled water devoid of electrolytes) or withpotable water (comprising electrolytes). Composition 8a Days of withwater for injection with potable water storage at API content API PurityAPI content API Purity 5° C. [% LC] [%] [% LC] [%] 0 96.0 99.9 96.0 99.917 96.0 99.8 96.0 99.9 25 96.0 99.8 92.0 99.9 31 92.0 99.9 92.0 99.9

1. A pharmaceutical composition comprising a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹ is hydrogen orC₁₋₇-alkyl; R² is hydrogen, cyano, C₁₋₇-haloalkyl or C₃₋₈-cycloalkyl; R³is hydrogen, C₁₋₇-alkyl, or C₃₋₈-cycloalkyl; A is N-heterocycloalkyl orNR¹²R¹³, wherein N-heterocycloalkyl comprises 1 or 2 nitrogen ring atomsand is optionally substituted with 1, 2, 3 or 4 substituents selectedfrom R¹⁴; R¹² is heterocycloalkyl comprising 1 nitrogen ring atom,wherein heterocycloalkyl is optionally substituted with 1, 2, 3 or 4substituents selected from R¹⁴; R¹³ is hydrogen, C₁₋₇-alkyl orC₃₋₈-cycloalkyl; R¹⁴ is independently selected from hydrogen,C₁₋₇-alkyl, amino, amino-C₁₋₇-alkyl, C₃₋₈-cycloalkyl andheterocycloalkyl or two R¹⁴ together form C₁₋₇-alkylene; with theproviso that if A is N-heterocycloalkyl comprising only 1 nitrogen ringatom, then at least one R¹⁴ substituent is amino or amino-C₁₋₇-alkyl;wherein the composition is an oral aqueous solution or a dry powdersuitable for constitution of an oral aqueous solution.
 2. Thepharmaceutical composition according to claim 1, or a pharmaceuticallyacceptable salt thereof, wherein R¹ is hydrogen or C₁₋₇-alkyl; R² ishydrogen, cyano, C₁₋₇-haloalkyl or C₃₋₈-cycloalkyl; R³ is hydrogen,C₁₋₇-alkyl, or C₃₋₈-cycloalkyl; A is N-heterocycloalkyl comprising 1 or2 nitrogen ring atoms, wherein N-heterocycloalkyl is optionallysubstituted with 1, 2, 3 or 4 substituents selected from R¹⁴; R¹⁴ isindependently selected from hydrogen, C₁₋₇-alkyl, amino,amino-C₁₋₇-alkyl, C₃₋₈-cycloalkyl and heterocycloalkyl or two R¹⁴together form C₁₋₇-alkylene; with the proviso that if A isN-heterocycloalkyl comprising only 1 nitrogen ring atom, then at leastone R¹⁴ substituent is amino or amino-C₁₋₇-alkyl.
 3. The pharmaceuticalcomposition according to claim 1, or a pharmaceutically acceptable saltthereof, wherein A is

wherein X is N or CH; R⁴ is hydrogen, C₁₋₇-alkyl or —(CH₂)_(m)—NR⁹R¹⁰;R⁵ is hydrogen or C₁₋₇-alkyl; R⁶ is hydrogen or C₁₋₇-alkyl; R⁷ ishydrogen or C₁₋₇-alkyl; R⁸ is hydrogen or C₁₋₇-alkyl; R⁹ and R¹⁰ areindependently selected from hydrogen, C₁₋₇-alkyl and C₃₋₈-cycloalkyl;R¹³ is hydrogen, C₁₋₇-alkyl or C₃₋₈-cycloalkyl; n is 0, 1 or 2; m is 0,1, 2 or 3; or R⁴ and R⁵ together form C₁₋₇-alkylene; or R⁴ and R⁷together form C₁₋₇-alkylene; or R⁵ and R⁶ together form C₂₋₇-alkylene;or R⁵ and R⁷ together form C₁₋₇-alkylene; or R⁵ and R⁹ together formC₁₋₇-alkylene; or R⁷ and R⁸ together form C₂₋₇-alkylene; or R⁷ and R⁹together form C₁₋₇-alkylene; or R⁹ and R¹⁰ together form C₂₋₇-alkylene;with the proviso that if X is CH then R⁴ is —(CH₂)_(m)—NR⁹R¹⁰; and withthe proviso that if X is N and R⁴ is —(CH₂)_(m)—NR⁹R¹⁰ then m is 2 or 3.4. The pharmaceutical composition according to claim 1, or apharmaceutically acceptable salt thereof, wherein A is selected from thegroup of:

wherein R¹¹ is hydrogen or C₁₋₇-alkyl.
 5. The pharmaceutical compositionaccording to claim 1, or a pharmaceutically acceptable salt thereof,wherein A is selected from the group of:


6. The pharmaceutical composition according to claim 1, wherein thecompound of formula (I) is selected from the group consisting of:2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;7-(1,4-diazepan-1-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;7-(1,4-diazepan-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one;7-(3,3-dimethylpiperazin-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3 S,5S)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-(3,3-dimethylpiperazin-1-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(3 S,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(3R)-3-ethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;and pharmaceutically acceptable salts thereof.
 7. The pharmaceuticalcomposition according to claim 1, wherein the compound of formula (I) isselected from the group consisting of:7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;7-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;and pharmaceutically acceptable salts thereof.
 8. The pharmaceuticalcomposition according to claim 1, wherein the compound of formula (I) is7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneor a pharmaceutically acceptable salt thereof.
 9. The pharmaceuticalcomposition according to claim 1, wherein the compound of formula (I) is7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneor a pharmaceutically acceptable salt thereof.
 10. The pharmaceuticalcomposition according to claim 1, wherein the oral aqueous solution hasa pH of less than pH4.
 11. The pharmaceutical composition according toclaim 1, wherein the pharmaceutical composition further comprises acitrate, malate, maleate or tartrate buffer system; or alternativelytartaric acid.
 12. The pharmaceutical composition according to claim 1,wherein the composition further comprises a extragranular fillerselected from lactose, starch, hydrolyzed starch, maltodextrin,microcrystalline cellulose, mannitol, sorbitol, sucrose, dextrose,dibasic calcium phosphate, calcium sulfate, and combinations thereof.13. The pharmaceutical composition according to claim 1, wherein thecomposition further comprises a diluent selected from lactose, starch,hydrolyzed starch, maltodextrin, microcrystalline cellulose, mannitol,isomalt, sorbitol, sucrose, dextrose, dibasic calcium phosphate, calciumsulfate, and combinations thereof.
 14. The pharmaceutical compositionaccording to claim 1, wherein the composition further comprises adiluent selected from lactose, starch, hydrolyzed starch,microcrystalline cellulose, mannitol, sorbitol, sucrose, dextrose,dibasic calcium phosphate, calcium sulfate, and combinations thereof.15. The pharmaceutical composition according to claim 1, wherein thecomposition further comprises a preservative, stabilizer or antioxidantsselected from vitamin A, vitamin C, vitamin E, vitamin E TPGS, retinylpalmitate, selenium, cysteine, methionine, citric acid, sodium citrate,methyl paraben, propyl paraben, disodium edetate, butyl hydroxyl toluol,riboflavin, ascorbic acid and combinations thereof.
 16. Thepharmaceutical composition according to claim 1, wherein the compositionfurther comprises a preservative selected from sorbic acid and sodiumbenzoate.
 17. The pharmaceutical composition according to claim 1,wherein the composition further comprises an antioxidant selected fromascorbic acid.
 18. The pharmaceutical composition according to claim 1,wherein the composition further comprises a stabilizer selected fromdisodium ethylenediaminetetraacetate.
 19. The pharmaceutical compositionaccording to claim 1, wherein the composition further comprises alubricant selected from poly(ethylene glycol).
 20. The pharmaceuticalcomposition according to claim 1, wherein the composition comprises: 1to 10% wt of a compound of formula (I) or a pharmaceutically acceptablesalt thereof; 5 to 15% wt of a buffer system or alternatively thecorresponding acid of a buffer system alone; 40 to 70% wt of a diluent;1 to 4% wt of an antioxidant; 0.5 to 2% wt of a stabilizer; 0.5 to 2% wof a lubricant; 1 to 8% wt of a preservative, 0 to 3% wt of a sweetener;and 0 to 20% wt of a flavor; and wherein the total amount of ingredientsdoes not exceed 100% wt.
 21. The pharmaceutical composition according toclaim 1, wherein the composition comprises: 1 to 10% wt of7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-oneor a pharmaceutically acceptable salt thereof; 5 to 15% wt of a buffersystem selected from citrate, malate, maleate or tartrate oralternatively tartaric acid alone; 40 to 70% wt of a diluent selectedfrom mannitol or a mixture of mannitol and isomalt; 1 to 4% wt ofascorbic acid; 0.5 to 2% wt of disodium edetate; 0.5 to 2% w of PEG6000;1 to 8% wt of a preservative selected from sorbic acid or sodiumbenzoate, 0 to 3% wt of a sweetener selected from sucralose or sodiumsaccharin; and 0 to 20% wt of a flavor selected from strawberry flavoror vanilla flavor; and wherein the total amount of ingredients does notexceed 100% wt.
 22. The pharmaceutical composition according to claim 1,wherein the composition is an oral aqueous solution.
 23. Thepharmaceutical composition according to claim 1, wherein the compositionis dry powder suitable for constitution of an oral aqueous solution. 24.A kit for the preparation of pharmaceutical compositions comprising thedry powder of claim 23 and water as solvent for constitution. 25.-29.(canceled)
 30. A pharmaceutical composition according to claim 1 furthercomprising olesoxime.
 31. The pharmaceutical composition according toclaim 30 further comprising an oil selected from sesame oil, olive oil,soya oil, cotton oil, castor oil, nut oil, rapeseed oil, corn oil,almond oil, sunflower oil, and combinations thereof.
 32. Thepharmaceutical composition according to claim 31 further comprising anemulsifying and/or lipophilic solubilizing agent selected from glycerylmono-oleate, glyceryl mono-linoleate, sorbitan mono-oleate, oleic acid,and combinations thereof; and optionally a polar surfactant.
 33. Thepharmaceutical composition according to claim 33 wherein the polarsurfactant is selected from polysorbate 80, caprylocaproyl polyoxylglycerides, and combinations thereof.
 34. A kit for the preparation ofpharmaceutical compositions comprising: a compound of formula (I)according to claim 1 or a pharmaceutically acceptable salt thereof; apowder blend as vehicle suitable for constitution of the compound offormula (I) or a pharmaceutically acceptable salt thereof; optionallywater as solvent for constitution; olesoxime; an oil; an emulsifyingand/or lipophilic solubilizing agents; and optionally a polarsurfactant. 35.-36. (canceled)
 37. A method for the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, which methodcomprises administering a pharmaceutical composition according to claim1 to a subject. 38.-51. (canceled)
 52. A method for the treatment,prevention, delaying progression and/or amelioration of diseases causedby an inactivating mutation or deletion in the SMN1 gene and/orassociated with loss or defect of SMN1 gene function, and/or for theprotection of cells implicated in the pathophysiology of the disease,which method comprises administering a combination of a compound offormula (I) according to claim 1, or a pharmaceutically acceptable saltthereof and olesoxime to a subject. 53.-57. (canceled)